Design, synthesis, and evaluation of orally active 4-(2,4-difluoro-5- (methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]triazines as dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 inhibitors

Article abstract of DOI:10.1021/jm0501275

A series of substituted 4-(2,4-difluoro-5-(methoxycarbamoyl)phenylamino) pyrrolo[2,1-f][1,2,4]-triazines was identified as potent and selective inhibitors of the tyrosine kinase activity of the growth factor receptors VEGFR-2 (Flk-1, KDR) and FGFR-1. The enzyme kinetics associated with the VEGFR-2 inhibition of compound 50 (K_i = 52 ± 3 nM) confirmed that the pyrrolo-[2,1-f][1,2,4]triazine analogues are competitive with ATP. Several analogues demonstrated low-nanomolar inhibition of VEGF- and FGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation. Replacement of the C6-ester substituent of the pyrrolo[2,1-f][1,2,4]-triazine core with heterocyclic bioisosteres, such as substituted 1,3,5-oxadiazoles, afforded compounds with excellent oral bioavailability in mice (i.e., 50 F_po = 79%). Significant antitumor efficacy was observed with compounds 44, 49, and 50 against established L2987 human lung carcinoma xenografts implanted in athymic mice. A full account of the synthesis, structure-activity relationships, pharmacology, and pharmacokinetic properties of analogues within the series is presented.

Full text of DOI:10.1021/jm0501275

J. Med. Chem. 2005, 48, 3991-4008
3991
Design, Synthesis, and Evaluation of Orally Active
4-(2,4-Difluoro-5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]triazines
as Dual Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth
Factor Receptor-1 Inhibitors
Robert M. Borzilleri,*,† Xiaoping Zheng,† Ligang Qian,† Christopher Ellis,† Zhen-wei Cai,† Barri S. Wautlet,‡
Steve Mortillo,‡ Robert Jeyaseelan, Sr.,‡ Daniel W. Kukral,‡ Aberra Fura,§ Amrita Kamath,§ Viral Vyas,§
John S. Tokarski,| Joel C. Barrish,† John T. Hunt,‡ Louis J. Lombardo,† Joseph Fargnoli,‡ and Rajeev S. Bhide†
Departments of Oncology Chemistry, Discovery Biology, Metabolism and Pharmacokinetics, and Structural Biology and
Modeling, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000
Received February 9, 2005
A series of substituted 4-(2,4-difluoro-5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]-
triazines was identified as potent and selective inhibitors of the tyrosine kinase activity of the
growth factor receptors VEGFR-2 (Flk-1, KDR) and FGFR-1. The enzyme kinetics associated
with the VEGFR-2 inhibition of compound 50 (Ki ) 52 ( 3 nM) confirmed that the pyrrolo-
[2,1-f][1,2,4]triazine analogues are competitive with ATP. Several analogues demonstrated low-
nanomolar inhibition of VEGF- and FGF-dependent human umbilical vein endothelial cell
(HUVEC) proliferation. Replacement of the C6-ester substituent of the pyrrolo[2,1-f][1,2,4]-
triazine core with heterocyclic bioisosteres, such as substituted 1,3,5-oxadiazoles, afforded
compounds with excellent oral bioavailability in mice (i.e., 50 Fpo ) 79%). Significant antitumor
efficacy was observed with compounds 44, 49, and 50 against established L2987 human lung
carcinoma xenografts implanted in athymic mice. A full account of the synthesis, structure-
activity relationships, pharmacology, and pharmacokinetic properties of analogues within the
series is presented.
Introduction
VPF),6 a key stimulator of tumor angiogenesis, promotes
endothelial cell proliferation, survival, migration, inva-
sion, and differentiation. The intrinsic activity respon-
sible for these cellular events is dependent on the highly
specific binding of the VEGF ligands (isoforms) to their
respective cell surface expressed receptors VEGFR-1
(Flt-1), VEGFR-2 (KDR, Flk-1), and VEGFR-3 (Flt-4).
Activation of the VEGF receptor tyrosine kinases (RTKs),
which are highly expressed on vascular endothelial cells,
leads to receptor dimerization, autophosphorylation of
key tyrosine residues within the catalytic domain, and
the initiation of downstream signaling cascades that
regulate tumor progression and dissemination.
Throughout the past decade, RTKs have received
considerable attention as attractive molecular targets
for drug discovery.7,8 Monoclonal antibodies specific for
kinase ligands or their receptors, and small molecule
inhibitors that interact at the ATP-binding site of a
given kinase domain have demonstrated promising
therapeutic utility.9 For example, the chimeric anti-
VEGF monoclonal antibody bevacizumab (Genentech)
recently afforded a significant survival benefit in meta-
static colorectal cancer patients when combined with
standard chemotherapy (IFL or Saltz regimen).10 These
key clinical findings provided the proof-of-concept for
therapies aimed at controlling abnormal vascular de-
velopment operative in cancer progression. Orally ac-
tive, small molecule inhibitors of VEGFR-2 kinase
activity from several structural classes11 (i.e., phthala-
zines,12 indolinones,13 quinazolines,14 isothiazoles,15a
thienopyridines,15b indeno[2,1-R]pyrrolo[3,4-c]carbazo-
lones,16 thiazoles,17a pyridines,17b indoles,17c,d imidazopyri-
Angiogenesis is an essential series of molecular events
that occurs during the formation of new blood vessels
from the endothelium of preexisting vasculature.1 This
tightly regulated process is dependent on a delicate
balance between pro- and antiangiogenic factors,2 and
serves as an integral component for physiological pro-
cesses, such as embryonic development, female repro-
ductive function, and wound healing. Uncontrollable or
pathological angiogenesis, however, has been implicated
in a wide array of vascular hyperproliferative disorders,
most notably cancer, rheumatoid arthritis, diabetic
retinopathy, age-related macular degeneration, and
psoriasis.3-4 It is well established that an efficient blood
supply is required for solid tumors to sustain growth
beyond a critical size and to metastasize to other organs.
Tumor-induced angiogenesis is responsible for the newly
formed capillary networks between host and tumor that
allow for effective delivery of oxygen and essential
nutrients to cancer cells, elimination of waste materials
generated from tumor metabolism, and migration of
cancer cells to remote sites leading to metastasis.5
Tumor cells respond to a variety of stimuli, such as
hypoxia, low pH, changes in interstitial fluid pressure,
growth factors, or cytokines by up-regulating the ex-
pression of proangiogenic factors. Vascular endothelial
growth factor (VEGF; vascular permeability factor,
* To whom correspondence should be addressed. Phone: (609) 252-
6236. Fax: (609) 252-7410. E-mail: robert.borzilleri@bms.com.
† Department of Oncology Chemistry.
‡ Department of Discovery Biology.
§ Department of Metabolism and Pharmacokinetics.
| Department of Structural Biology and Modeling.
10.1021/jm0501275 CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/17/2005

3992 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
Chart 1. Examples of VEGFR-2 Inhibitors Undergoing
Clinical Trials
Figure 1. Evolution of the hydroxamate series of VEGFR-2
inhibitors.
since antiangiogenic therapies are directed toward
highly regulated endothelial cells of the host, rather
than genetically unstable tumor cells, it is anticipated
that selective VEGFR-2 and/or FGFR-1 inhibition will
lead to cancer therapeutics that have a lower risk of
developing classical drug resistance compared to tradi-
tional chemotherapy.22
As part of our continuing effort to identify selective
small molecule RTK inhibitors for various oncology
indications, we have identified the pyrrolo[2,1-f][1,2,4]-
triazine nucleus as a general kinase inhibitor tem-
plate.23 In our search for selective VEGFR-2 kinase
inhibitors, we have recently disclosed the structure-
activity relationships (SARs) of 4-(3-hydroxy-4-meth-
ylphenylamino)pyrrolo[2,1-f][1,2,4]triazines 2a (Figure
1).24a Herein, we describe the SAR, pharmacokinetics,
and pharmacology of the novel series of 4-(2,4-difluoro-
5-(methoxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]-
triazines 2b that were derived from the phenol ana-
logues and a structurally related series of p38 MAP
kinase inhibitors.24b These studies culminated in the
identification of potent, orally active dual inhibitors of
VEGFR-2 and FGFR-1 from the hydroxamate series
that demonstrated robust antitumor activity in the
L2987 human lung carcinoma xenograft model.
dines,17e benzimidazoles,17f pyrimidines,17g-i diamino-
[1,3,5]triazines,18a biheteroaryls18b) have also progressed
to various stages of preclinical and clinical development.
Representative examples of VEGFR-2 inhibitors cur-
rently undergoing clinical evaluation are illustrated in
Chart 1. Novartis’ anilinophthalazine PTK-787 (vata-
lanib, 1a)12a,b and Pfizer’s (Sugen/Pharmacia) third-
generation indolinone SU-11248 (1b)13a,b are the most
advanced agents, currently in phase III clinical trials.
AstraZeneca and Pfizer have initiated phase II clinical
trials for quinazoline ZD-6474 (vandetanib, 1c)14a,b and
isothiazole CP-547632 (1e),15a respectively. The inde-
nopyrrolocarbazole N,N-dimethylglycine ester prodrug
CEP-705516 (1f, Cephalon) and a second-generation
quinazoline AZD-2171 (1d, AstraZeneca) have also
entered the clinic. Each of these structurally diverse
chemotypes offer distinct kinase selectivity profiles
relative to VEGFR-2 activity that may ultimately influ-
ence their overall therapeutic potential.
Fibroblast growth factor (FGF) represents another
important family of angiogenic growth factors.19 While
the exact role of FGF in tumor angiogenesis remains
elusive, this potent mitogen of vascular endothelial cells
and fibroblasts is believed to cooperate with VEGF
during blood vessel development.20 Since VEGFR-2 and
FGFR-1 have been shown to directly influence tumor
angiogenesis, simultaneous inhibition of these two RTKs
is postulated to enhance antitumor activity. To this end,
small molecule inhibitors of FGFR-1,21 including indoli-
none derivatives13c that inhibit VEGFR-2, FGFR-1, and
PDGFR-â have appeared in the literature. Furthermore,
Chemistry
Pyrrolo[2,1-f][1,2,4]triazine derivatives 8-18 were
prepared according to the general synthetic sequence
outlined in Scheme 1. The pyrrolotriazinone core 5 was
assembled using a modified version of the literature
procedure of Patil et al.25 N-Amination of diethyl 3-iso-
propyl-1H-pyrrole-2,4-dicarboxylate (3)26 using either
O-(diphenylphosphinyl)hydroxylamine or O-(mesitylene-
sulfonyl)hydroxylamine provided aminopyrrole 4. Cy-
clization of 4 upon heating in formamide generated the
desired bicycle 5. Treatment of intermediate 5 with
phosphorus oxychloride afforded chloroimidate 6, which
was subsequently coupled with the appropriately func-
tionalized 3-hydroxy or 3-amino-N-alkoxybenzamides 7
to furnish the corresponding analogues 8-18 in good
overall yield.
A small library of C6-carbamate analogues (cf. Sup-
porting Information) was prepared from the stable acyl
azide intermediate 20 using a parallel, solution phase
format (Scheme 2). Thus, carboxylic acid 19, derived
from base-promoted hydrolysis of ethyl ester 14, was
treated with diphenylphosphoryl azide (DPPA) in the
presence of triethylamine to furnish compound 20.
Curtius rearrangement of the acyl azide 20, in the
presence of commercially available alcohols, produced
the desired carbamate derivatives, including examples
21-26.
Five-membered ring heterocycles were installed at the
6-position of the pyrrolo[2,1-f][1,2,4]triazine nucleus

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3993
Scheme 1a
a Reagents and conditions: (a) Ph2PO2NH2 or Me3PhSO3NH2, NaH, DMF, 85%; (b) formamide, 165 °C, 90%; (c) POCl3, 110 °C, 3 h,
90%; (d) X ) O: K2CO3, DMF, 25 °C, 18 h, X ) NH: DMF, 25 °C, 12-18 h.
Scheme 2a
a Reagents and conditions: (a) 1 N NaOH, THF-H2O, 60 °C, 27 h, 98%; (b) DPPA, 1,4-dioxane, Et3N, 40 °C, 1 h, 99%; (c) R2OH, DMF,
85 °C, 2 h, 50-65%.
Scheme 3a
a Reagents and conditions: (a) 1 N NaOH, THF-H2O, 60 °C, 98%; (b) 1-aminopropan-2-one, BOP reagent, Et3N, DMF, 50 °C, 2 h, 61%;
(c) POCl3, 120 °C, 6 h; (d) 5-amino-2,4-difluoro-N-methoxybenzamide, MeCN, 25 °C, 18 h; (e) (1) N-hydroxyacetamidine, TBTU, cat. HOBt,
i-Pr2NEt, DMF, 1.5 h, (2) DMF, 140 °C, 3 h, 57% overall.
according to the synthetic sequences illustrated in
Schemes 3-5.27-32 As shown in Scheme 3, the pyrrolo-
triazinone ester 5 was hydrolyzed under mild heating
to provide carboxylic acid 27. Oxazole 28 and 1,2,4-
oxadiazole 30 were prepared from intermediate 27 by
two independent routes. Standard amide bond coupling
of compound 27 with 1-aminopropan-2-one was ac-
complished with benzotriazol-1-yloxy-tris(dimethylami-
no)phosphonium hexafluorophosphate (BOP). The amide
intermediate was heated with phosphorus oxychloride
to promote simultaneous cyclization and chloroimidate
formation to furnish 28. Alternatively, acid 27 was
coupled to N-hydroxyacetamidine in the presence of
O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroni-
um tetrafluoroborate (TBTU) and then cyclized under
thermal conditions to generate 30 in good overall yield.
Treatment of compound 28 and the chloroimidate
derived from pyrrolotriazinone 30 with 5-amino-2,4-
difluoro-N-methoxybenzamide provided the fully elabo-
rated analogues 29 and 31, respectively. The regioiso-
meric 1,2,4-oxadiazole 34 was prepared from the
chloroimidate obtained from pyrrolotriazinone 33 using
similar chemistry (Scheme 4). The nitrile 32, derived
from carboxylic acid 27, was converted to the 1,2,4-
oxadiazole 33 using an unoptimized, two-step procedure
involving the cyclization of the requisite N-hydroxy-
acetamidine intermediate with acetyl chloride in re-
fluxing pyridine.

3994 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
Scheme 4a
a Reagents and conditions: (a) (1) NH2OH-HCl, K2CO3, EtOH reflux, 28 h, (2) AcCl, pyridine reflux, 7 h, 11% overall; (b) POCl3, 120
°C, 5 h; (c) 5-amino-2,4-difluoro-N-methoxybenzamide, MeCN, 25 °C, 14 h; 30% from 33.
Scheme 5a
a Reagents and conditions: (a) RCO2H, POCl3, 100 °C, 1 h then 120 °C, 3 h; (b) 5-amino-2,4-difluoro-N-methoxybenzamide, DMF, 50
°C, 1-6 h; (c) LiN[Si(CH3)3]2, (PhSO2)2NF, THF, -78 to -30 °C, 1 h, 65%; (d) acetamidine-HCl, DMF, 120 °C, 1 h, 86%; (e) POCl3, 120
°C, 5 h; (f) 71% overall yield from 51.
Table 1. Effect of the C4-Substituent on Enzymatic Activitya
The hydrazide intermediate 35, obtained from the
reaction of ester 5 with hydrazine, was used to prepare
1,3,4-oxadiazoles 37-49 and triazole 52 (Scheme 5). The
C6-heterocycles of compounds 36 and 51 were installed
under thermal conditions, using either the correspond-
ing carboxylic acid in phosphorus oxychloride or aceta-
midine hydrochloride in DMF. Alternatively, the oxa-
diazole rings of compounds 38 and 47 were prepared
from hydrazide 35 using triethyl orthoformate30 and
phosgene imminium chloride,33 respectively (cf. Experi-
mental Section). Chemistry described previously was
used to convert compounds 36 and 51 to the desired
VEGFR-2 inhibitors 37-49 and 52, respectively. Di-
fluorination of compound 49 was carried out at low
temperature in THF using excess lithium bis(trimeth-
ylsilyl)amide and 2.5 equiv of commercially available
N-fluorobenzenesulfonimide to furnish the desired ox-
azole analogue 50 in 65% yield.
IC50 (nM) for
VEGFR-2
compd
X
R1
R2
R3
kinase inhibnb
8
9
O
H
H
H
H
H
F
H
H
Me
Br
F
Me
Me
Me
Me
Me
Me
Me
Et
270
130
120
270
79
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
10
11
12
13
14
15
16
17
18
H
F
34
F
13
F
F
40
F
F
i-Pr
350
1200
190
F
F
i-Bu
F
F
CH2-cycc
Results and Discussion
a See Experimental Section for a description of the assay
conditions. b IC50 values are reported as the mean of at least three
determinations. Variability around the mean value was <25%.
c cyc ) cyclopropyl.
In Vitro Pharmacology and SAR. The pyrrolo[2,1-
f][1,2,4]triazine analogues depicted in Tables 1-4 were
initially screened and optimized for inhibition of human
VEGFR-2 tyrosine kinase activity. Potent analogues
(IC50 < 100 nM) identified in the primary biochemical
assay23 were then evaluated for inhibition of FGFR-1
kinase activity. Select compounds were subsequently
screened for enzyme inhibition against a small panel
of RTKs [Flk-1, PDGFR-â, HER-1 (EGFR), HER-2, IGF-
1R] utilizing a previously reported assay format.23 In
each case, the entire cytoplasmic domain sequence of
the RTK was fused to glutathione S-transferase and the
biological activity was determined by quantitation of the
amount of radioactive phosphate transferred to an
artificial poly(Glu4/Tyr) substrate. Procedures for mea-
suring activity against the representative nonreceptor
kinase LCK and the serine-threonine kinases PKCR and
CDK2 have also been described previously.34,35 The most
potent and selective VEGFR-2 and FGFR-1 inhibitors
were evaluated for their ability to inhibit growth-factor-
stimulated cellular proliferation of human umbilical
vein endothelial cells (HUVECs) using either VEGF or
FGF as the mitogen.23 As a positive control for general
cytotoxicity, compounds evaluated in the HUVEC as-
says were also tested for their ability to inhibit the
proliferation of the L2987 human lung carcinoma cell
line. In all cases, the pyrrolotriazine analogues described

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3995
Table 2. Effect of C6-Carbamates on Enzymatic and Cellular
marizes the SAR of the C4-hydroxamate group with
respect to inhibition of VEGFR-2 kinase activity. Con-
sistent with our earlier observations in the phenol
series,24 the amine linker (X ) NH) provided compounds
that were slightly more potent against VEGFR-2 kinase
than the corresponding ether analogues (i.e., 9 versus
8). N-Methylation of the hydroxamate group of com-
pound 9 provided ethyl 5-isopropyl-4-(3-(methoxy-
(methyl)carbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]-
triazine-6-carboxylate (structure not shown), which was
>30-fold less potent in the biochemical assay. The
significant reduction in activity supports the hypothesis
that the hydroxamate-NH participates in hydrogen-
bonding interactions within the VEGFR-2 kinase do-
main (vide infra). Addition of a methyl substituent at
R2 (i.e., 10) had minimal effect on the intrinsic activity,
while the bromide analogue 11 (R2 ) Br) was >2-fold
less potent than the parent hydroxamate derivative 9.
Incorporation of a fluorine atom at R2 was well-toler-
ated, although the fluorine atom of analogue 13 (R1 )
F) had a greater impact on the VEGFR-2 kinase activity
compared to compound 12. The 2,4-difluoro analogue
14 was one order of magnitude more potent than the
original lead 9 in the kinase assay and displayed
excellent cellular potency (IC50 ) 4.8 nM) in the VEGF-
driven HUVEC proliferation assay. Increasing the size
of hydroxamate substituent R3 (i.e., 15-18) was detri-
mental to the kinase activity. Since an electron-deficient
phenyl ring provides a significant enhancement in the
VEGFR-2 kinase inhibition, the 2,4-difluoro-5-(meth-
oxycarbamoyl)phenylamino group was selected as the
preferred C4-substituent for subsequent SAR studies.
Activitya
a See Experimental Section for a description of the assay
conditions. b IC50 values are reported as the mean of at least three
determinations. Variability around the mean value was <25%.
c Human umbilical vein endothelial cells. d ND ) not determined.
Table 3. Comparison of VEGFR-2 and FGFR-1 Biochemical
and Cellular Potenciesa for C6-Heterocyclic Analogues
The C6-carbamate analogues obtained from the li-
brary synthesis were initially screened for VEGFR-2
kinase inhibition at concentrations of 0.2 and 0.05 µM
(cf. Supporting Information). The IC50 values were then
determined for select compounds of interest, which
provided g50% inhibition at 0.05 µM in the preliminary
two-point assay. Table 2 highlights representative car-
bamate analogues from the library that offer different
physicochemical properties and a range of in vitro rates
of metabolism in human liver microsomes (vide infra).
In contrast to the SAR observed with the phenol series,24
replacement of the metabolically labile C6-ester moiety36
of compound 14 with a carbamate group provided
pyrrolotriazine analogues that were consistently less
potent against VEGFR-2 kinase. For example, carbam-
ates 21 and 22 were 5- and 14-fold less potent than ester
analogue 14, respectively. On the basis of the proposed
binding mode of the pyrrolo[2,1-f][1,2,4]triazines in the
VEGFR-2 kinase domain, the C6-substituents extend
out toward the protein surface and potentially interact
with solvent. Thus, appending a terminal piperidine or
methyl sulfone substituent to the carbamate via a
3-carbon linker was found to be well-tolerated (i.e., 23
and 24). Tetrahydrofuran analogue 25 provided com-
parable enzymatic activity to compound 21, whereas the
N-methylpiperazine derivative 26 was the most potent
carbamate analogue in both the kinase and cellular
proliferation assays, providing IC50 values of 36 and 19
nM, respectively. As shown in Table 2, in most cases
there is good agreement between the IC50 values ob-
tained in the VEGFR-2 kinase and VEGF-stimulated
HUVEC proliferation assays for the carbamate series.
a See Experimental Section for a description of the assay
conditions. b IC50 values are reported as the mean of at least three
determinations. Variability around the mean value was <50%.
c Human umbilical vein endothelial cells. d ND ) not determined.
herein provided IC50 values >1.3 µM in the unstimu-
lated growth of L2987 cells.
The pyrrolo[2,1-f][1,2,4]triazine nucleus with the
C5-isopropyl and C6-ethyl ester substituents served as
the starting template for the hydroxamate series of
VEGFR-2 kinase inhibitors based on our previous SAR
investigations involving the 4-(3-hydroxy-4-methyl-
phenylamino)pyrrolo[2,1-f][1,2,4]triazines.24 Table 1 sum-

3996 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
Table 4. VEGFR-2 and FGFR-1 Biochemical and Cellular
compound 46 demonstrated an order of magnitude
decrease in cellular potency against VEGF- and FGF-
stimulated HUVECs. Gradually increasing the electron-
withdrawing capacity (higher σ value) of the R-substit-
uent on the oxadiazole ring, as in the case of compounds
44 and 45, provided a proportional decrease in biochemi-
cal potency and growth inhibition in the HUVEC
proliferation assay. Alternatively, introduction of an
electron-donating group, such as the dimethylamino
substituent of compound 47, led to improved potency
in both the kinase and HUVEC proliferation assays. In
an effort to optimize the ADME properties and enhance
the aqueous solubility of the series, polar substituents,
such as the dimethylaminomethyl and methylsulfonyl-
methyl groups, were appended to the oxadiazole core.
Amine analogue 48 was less potent than methyloxadia-
zole 37 in the kinase assays; however, the sulfone
derivative 49 afforded excellent biochemical and cellular
potency. Consistent with previous results, the more
electron deficient difluorosulfone oxadiazole 50 demon-
strated comparable VEGFR-2 and FGFR-1 kinase activ-
ity to that of analogue 44 with IC50 values of 53 and
220 nM, respectively. The cellular behavior of compound
50 in the VEGF- and FGF-simulated HUVEC prolifera-
tion assays was consistent with the biochemical data
obtained for the two enzymes. To better understand the
enzyme inhibitory properties of 50, the Ki value was
determined for VEGFR-2 kinase. Oxadiazole 50 was
confirmed to be an ATP-competitive inhibitor of VEGFR-
2, with an apparent Ki value of 52 ( 3 nM. The ability
of the VEGFR-2 and FGFR-1 proteins to accommodate
the diverse set of R groups illustrated in Table 4 again
suggests that the C6-oxadiazole ring and appended
substituents occupy a more flexible portion of the
receptor that extends out toward solvent.
Proposed Binding Model for Compound 50. To
better understand the binding mode of the 4-(5-(meth-
oxycarbamoyl)phenylamino)pyrrolo[2,1-f][1,2,4]-
triazines, a binding model of oxadiazole 50 in the ATP
site of VEGFR-2 was developed using a reported crystal
structure of the VEGFR-2 kinase domain.37 Compound
50 was docked into the VEGFR-2 structure using the
GLIDE software38 and then energy-minimized with the
CFF force field for 2000 steps of conjugate gradient
minimization.39 VEGFR-2 residues within 6 Å of the
ligand were allowed to relax. Figure 2 depicts the
proposed binding mode of 50 in the ATP binding pocket.
A highly conserved hydrogen-bond interaction be-
tween the backbone amide-NH of Cys919 and the N1
nitrogen of the pyrrolo[2,1-f][1,2,4]triazine ring anchors
the inhibitor to the hinge region of the adenine binding
pocket. The aniline portion of the inhibitor is oriented
into a mostly hydrophobic pocket, deep within the
binding site formed by residues such as Val916 and
Leu889. The carbonyl and NH groups of the hydroxam-
ate moiety form critical hydrogen bonds with the
backbone NH of Asp1046 and the side chain of Glu885,
respectively. The C5-isopropyl group makes van der
Waals contacts with a hydrophobic cleft created by the
proximal Val848 and Leu1035 residues. These same
residues along with Cys1045 and Leu840 (not labeled
in figure) roughly define the pocket occupied by the
ribose ring of ATP. The oxadiazole heterocycle, while
nearly coplanar with the pyrrolo[2,1-f][1,2,4]triazine
Potenciesa of C6-Oxadiazole Analogues
IC50 (nM)
growth
inhibition
kinase inhibitionb
of HUVECsc
compd
R
VEGFR-2 FGFR-1 VEGF FGF
38
39
40
41
42
43
44
45
46
47
48
49
50
H
77
17
22
23
11
17
57
76
29
11
66
16
53
160
28
91
1.8
18
2.1
3.6
4.0
17
54
4.7
1.0
6.6
5.3
9.2
21
33
44
7.3
ND
4.6
Et
i-Pr
i-Bu
39
33
cyclopropyl
CH2-cyclopropyl
CHF2
14
27
100
84
CF3
35
25
CH2CF3
68
18
149
16
220
N(CH3)2
6.7
CH2N(CH3)2
CH2SO2CH3
CF2SO2CH3
NDd
2.1
27
130
a See Experimental Section for a description of the assay
conditions. b IC50 values are reported as the mean of at least three
determinations. Variability around the mean value was <50%.
c Human umbilical vein endothelial cells. d ND ) not determined.
It is well-documented in the literature that five-
membered heteroaromatic rings, such as oxazoles and
oxadiazoles, serve as excellent, hydrolytically stable
bioisosteres for an ester substituent.29,30 As shown in
Table 3, a series of methyl-substituted five-membered
ring heterocycles was identified as acceptable replace-
ments for the C6-ester or acid functionality of pyrrolo-
[2,1-f][1,2,4]triazine 14. Oxazole 29 and 1,3,4-oxadiazole
37 demonstrated essentially equivalent VEGFR-2 ki-
nase inhibitory activity compared to ester 14, and both
compounds inhibited FGFR-1 kinase activity with IC50
values ca. 20 nM. The triazole 52 and the two regio-
isomeric 1,2,4 oxadiazole analogues 31 and 34 were less
potent than 29 and 37 in the biochemical assays. The
cellular potencies of 52 in the VEGF or FGF-driven
HUVEC proliferation assays were consistent with the
enzymatic activities obtained in the corresponding ki-
nase assays. Compounds 29, 31, and 37 were signifi-
cantly more potent inhibitors of HUVEC proliferation,
regardless of the mitogen employed (IC50 < 10 nM).
Although the in vitro pharmacology of 29 was impres-
sive, the oxazole analogue demonstrated significant
activity against a panel of cytochrome (CYP) P450
isozymes (IC50 values: CYP1A2, 0.064 µM; CYP2C9, 3.6
µM; and CYP3A4, 5.1 µM).
Since compound 37 provided an encouraging in vitro
biological profile and an efficient synthesis of the 1,3,4-
oxadiazole ring was available, the SAR of a series of
substituted C6-oxadiazole analogues was investigated
(Table 4). The unsubstituted oxadiazole 38 provided a
significant reduction in biochemical and cellular potency
compared to 37, whereas substitution of the oxadiazole
ring with larger aliphatic groups (i.e., 39-43) was well-
tolerated. Interestingly, while fluorination of the ethyl
substituent of oxadiazole 39 did not significantly affect
the kinase inhibitory activity for the two enzymes,

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3997
min provided metabolic rates of 0.30, 0.074, and 0.104
nmol/min/mg protein, respectively. Moderate to high
rates of drug conversion were obtained in HLM with
the oxadiazole derivatives, which suggests the potential
for high clearance in humans. Incubation of a 10 µM
concentration of the methyl sulfone oxadiazole 49 in
HLM for 10 min provided a high metabolic rate of 0.60
nmol/min/mg protein. However, the HLM values for the
fluorinated sulfone derivative 50 and the difluoromethyl
analogue 44 were ca. 2-fold lower (0.27 and 0.35 nmol/
min/mg protein, respectively), presumably due to re-
moval of a potential site of oxidative metabolism.
To quickly assess the preliminary pharmacokinetic
(PK) properties of this class of pyrrolo[2,1-f][1,2,4]-
triazines, several carbamate and oxadiazole analogues
were administered orally (50 mg/kg dose except for
compound 50) to male Balb/C mice and the plasma level
concentrations were recorded at 1 and 4 h time points
(Table 6). Carbamates 24-26 yielded relatively low drug
exposures during the 4 h study. In contrast, oxadiazole
analogues 37, 44, 49, and 50 were all well-absorbed,
producing remarkably high plasma level concentrations
at the 1 h time point. Oxadiazoles 44 and 49 maintained
high plasma exposures over the 4 h period, while the
drug concentrations of compounds 50 and 37 decreased
2- and 3.5-fold, respectively.
Further assessment of the pharmacokinetics associ-
ated with compound 50 in Balb/C mice revealed that
the steady-state volume of distribution (Vss) was greater
than total plasma/blood volume but less than total body
water. These results are indicative of moderate ex-
travascular distribution. The systemic plasma clearance
(Cl) of compound 50 was less than 2% of hepatic blood
flow. A 10 mg/kg oral dose administered as a solution
in 3:1:6 poly(ethylene glycol):ethanol:water was readily
Figure 2. Binding model of compound 50 in the ATP binding
site of VEGFR-2 kinase. Carbon atoms of 50 are in green and
carbon atoms of the protein are in gray. Potential hydrogen-
bond interactions are indicated by magenta dotted lines.
ring, points toward surface-exposed protein. Although
not ultimately required for activity, the sulfone oxygen
atoms likely form hydrogen bonds with the backbone
NH and side chain of Asn923. In general, the binding
model is consistent with the SAR and enzyme kinetics
described above for the hydroxamate series.
Kinase Selectivity Profile for Select Analogues.
The most promising carbamate and oxadiazole ana-
logues were screened against a representative panel of
kinases, which included RTKs, non-RTKs, and serine/
threonine kinases (Table 5). Since antiangiogenesis
therapies target endothelial cells (of the host) rather
than the tumor and preclinical efficacy studies are
conducted in mice, Flk-1, the mouse homologue of
human VEGFR-2 was also included in the panel.
Compared to their VEGFR-2 inhibitory activity, a
significant reduction in potency was observed for Flk-
1, particularly for carbamate 24 (24-fold) and oxadiaz-
oles 44 and 50 (8-15-fold). Although carbamate ana-
logues 24 and 25 demonstrated single-digit micromolar
activity against the HER-1 and HER-2 enzymes, the
oxadiazole derivatives exhibited >150-fold selectivity for
VEGFR-2 versus these epidermal growth factor receptor
family members. Both the carbamate and oxadiazole
analogues provided excellent VEGFR-2 kinase selectiv-
ity relative to other RTKs, such as PDGFR-â and IGF-
1R. While maintaining reasonable selectivity for VEG-
FR-2 and FGFR-1 kinases (33- and 25-fold, respectively),
oxadiazole 37 provided an IC50 ) 500 nM against the
non-RTK LCK. The other analogues depicted in Table
5 were considerably less potent against LCK. Similarly,
no significant activity was detected with any of the
hydroxamate-based, dual VEGFR-2 and FGFR-1 inhibi-
tors versus the serine/threonine kinases PKCR and
CDK2.
absorbed and demonstrated a favorable half-life (t1/2
)
and mean residence time (MRT). The measured oral
bioavailability (Fpo) of compound 50 in this study was
79%. The PK parameters from the comprehensive
mouse study are summarized in Table 7. The plasma
protein binding (PB) of compound 50 was calculated to
be 99.8 ( 0.04% and 98.8 ( 0.11% in mouse and human
serum, respectively, using the equilibrium dialysis
method (at 10 µM). Although the unbound (free) fraction
available in mice was predicted to be low (0.2%), the
favorable pharmacokinetic prolife of 50 and the high
plasma concentrations obtained with several analogues
in the 4 h oral exposure studies warranted the advance-
ment of these analogues into in vivo efficacy studies.
In Vivo Antitumor Activity. Four oxadiazole ana-
logues were evaluated for antitumor efficacy in a L2987
human lung carcinoma xenograft model (Table 8). An
active result in this study is defined as greater than 50%
tumor growth inhibition (TGI) over at least one tumor
volume doubling time (cf. Experimental Section). Com-
pounds 37, 44, 49, and 50 were administered orally for
10 consecutive days (qd × 10) to nude mice bearing
tumors staged to 100-150 mm3. Oxadiazole 37, the
In Vitro Metabolic Stability and Pharmacoki-
netics. Several C6-carbamate and C6-heterocyclic pyr-
rolo[2,1-f][1,2,4]triazines with suitable inhibitory activ-
ity in the kinase and cellular proliferation assays were
screened for metabolic stability in human liver mi-
crosomes (HLM). In general, the carbamate analogues
exhibited a wide range of rates in this in vitro system
(cf. Supporting Information). For example, incubation
of the methyl sulfone, tetrahydrofuran, and N-meth-
ylpiperazinecarbamates 24-26 in HLM at 3 µM for 10
most potent inhibitor of Flk-1 kinase activity (IC50
)
0.086 µM), was toxic at the 50 mg/kg/day dose level in
the L2987 model. However, administration of compound
37 at a 5-fold lower dose (10 mg/kg), twice daily for 20
consecutive days (2qd × 20) provided good in vivo
efficacy (79% TGI) in this model. Even with the signifi-

3998 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
Table 5. Kinase Selectivity Profile for Select Carbamate and Oxadiazole Analoguesa
enzyme inhibition IC50 (µM )
compd
VEGFR-2
Flk-1
FGFR-1
HER-1
HER-2
PDGFR-â
IGF-1R
LCK
PKCR
CDK2
24
25
37
44
50
0.062
0.058
0.018
0.057
0.053
1.5
0.058
0.046
0.019
0.10
4.2
5.5
>10
>10
>25
5.3
>10
8.0
>10
>25
>2.0
>2.0
>2.0
>2.0
>2.0
>25
>25
>25
>25
>25
5.1
4.7
0.50
18
7.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.27
0.086
0.84
0.42
0.22
a See Experimental Section for a description of assay conditions.
Table 6. Mouse Exposure Data Following Oral Administration
of Select Compounds
plasma levels (µM)a,b
compd
at 1 h
at 4 h
24c
25c
26c
37c
44c
49c
50d
7.9 ( 0.7
29.5 ( 15.4
5.1 ( 0.8
131.6 ( 45.7
187.4 ( 54.6
287.8 ( 14.2
26.6 ( 3.2
13.8 ( 2.1
2.9 ( 1.5
3.0 ( 0.9
37.7 ( 17.0
154.9 ( 63.1
231.6 ( 23.8
13.8 ( 1.0
a Average of three male mice. b Vehicle: 3:1:6 PEG400/EtOH/
water. c 50 mg/kg dose. d 10 mg/kg dose.
Figure 3. Antitumor activity of compound 50 against estab-
lished L2987 human tumor xenografts implanted subcutane-
ously in athymic mice. Arrows indicate dosing (qd × 10).
Table 7. Pharmacokinetic Properties of Compound 50 in
Micea,b
parameter
unit
iv dose
po dose
dose
Cmax
Tmax
AUCtot
t1/2
MRT
Cl
mg/kg
5
23.7
-
97.4
2.5
4.9
1.53
0.45
-
10
27.7
0.5
155
3.6
5.2
-
the dosing regimen. The higher doses required for
efficacy in this model with 44 and 50 may be attributed
to the reduced Flk-1 activity of these compounds (IC50
values of 0.84 and 0.42 µM) compared to oxazole 37 (IC50
) 0.086 µM).
µM
h
µM‚h
h
h
mL/min/kg
L/kg
%
Figure 3 illustrates the dose-dependent growth inhi-
bition of compound 50 in the L2987 tumor model. The
two highest dose levels of 100 and 150 mg/kg/day
provided similar antitumor activity (% TGI ) 88 and
94%, respectively) with nearly complete tumor stasis
throughout the dosing regimen (maximum efficacy).
Consistent with the mechanisms of antiangiogenic
therapy relative to VEGFR-2/FGFR-1 inhibition, tumor
growth again commenced once compound dosing was
terminated. Even at the low dose of 50 mg/kg/day,
compound 50 was active, providing 64% tumor growth
inhibition. A minimum effective dose for oxadiazole 50
was not established in this study.
Vss
Fpo
-
79
a Composite serum concentration-time profiles were con-
structed for the PK analysis. b Vehicle: 3:1:6 PEG400/ethanol/
water.
Table 8. Growth Inhibition for Oxadiazole Analogues against
Established L2987 Human Lung Carcinoma Xenografts
Implanted Subcutaneously in Athymic Mice
dose
compd
(mg/kg/day)a
% TGIb
37
44
50
50
100
150
60
100
50
100
150
toxicc
30.0
46.5
88.4
42.6
74.9
63.9
87.5
94.2
Conclusions
49
50
Incorporation of the 2,4-difluoro-5-(methoxycarbam-
oyl)phenylamino substituent at the C4-position of the
pyrrolo[2,1-f][1,2,4]triazine scaffold resulted in potent
and selective inhibitors of the VEGFR-2 and FGFR-1
kinases. In addition to the biochemical potency, many
analogues demonstrated significant antiproliferative
effects in growth factor-dependent HUVEC assays.
Critical to the discovery of orally bioavailable analogues
in the series were the SAR studies carried out at the
6-position of the pyrrolo[2,1-f][1,2,4]triazine nucleus. A
survey of various C6-heterocycles led to the identifica-
tion of substituted 1,3,5-oxadiazoles that served as
metabolically stable bioisosteres for the C6-ester sub-
stituent. Preliminary assessment of the pharmacoki-
netic properties associated with compounds in the series
revealed that the C6-carbamate analogues suffered from
poor absorption, while several C6-heterocycles provided
high exposures following oral administration in mice.
a Daily oral administration of compound for 10 consecutive days
using 7:1:2 PEG 400/ethanol/water as the vehicle. b Maximum
percent tumor growth inhibition over one tumor doubling time;
>50% TGI is considered active in this study. c Compound 37 was
active when dosed at 10 mg/kg/d on a 2qd × 20 schedule.
cant potency against Flk-1, the twice daily (2qd) dosing
requirement for compound 37 may be a result of a
considerable decline in plasma level concentrations
following each dose, as observed in the 4 h oral exposure
studies (vide supra). Analogues 44 and 49 exhibited
antitumor activity at doses of 150 and 100 mg/kg/day,
respectively, while compound 50, provided robust in vivo
efficacy at multiple dose levels. No overt toxicity (mor-
bidity or weight loss) was observed with oxadiazole
analogues 44, 49, and 50 at any dose level throughout

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3999
to room temperature. The resulting precipitate was filtered,
washed with water, and dried overnight to afford 5 (4.0 g, 90%)
as a white solid: Anal. RP-HPLC tR ) 1.56 min (column A,
Further evaluation of oxadiazole 50 in mice showed that
this compound possessed excellent oral bioavailability
and a good half-life. The favorable in vitro pharmacology
and pharmacokinetic properties exhibited by the oxa-
diazole analogues 44, 49, and 50 translated into sig-
nificant antitumor activity in the L2987 human lung
carcinoma xenograft model.
1
purity 99%); H NMR (500 MHz, DMSO-d6) δ 11.68 (s, 1H),
7.90 (s, 1H), 7.86 (d, 1H, J ) 4.0 Hz), 4.24 (q, 2H, J ) 7.1 Hz),
4.20 (m, 1H), 1.31 (d, 6H, J ) 7.2 Hz), 1.29 (t, 3H, J ) 7.1
Hz); 13C NMR (125 MHz, DMSO-d6) δ 163.8, 155.0, 140.4,
134.7, 124.5, 117.6, 114.1, 60.1, 24.3, 21.8, 14.6; HRMS for
C12H15N3O3 (M + H)+ calcd 250.1113, found 250.1102. Anal.
(C12H15N3O3) C, H, N.
Experimental Section
Ethyl 4-Chloro-5-isopropylpyrrolo[2,1-f][1,2,4]triazine-
6-carboxylate (6). A mixture of 5 (17.8 g, 71.5 mmol), POCl3
(12.0 g, 78.6 mmol), and a catalytic amount of Et3N (1.00 mL,
7.15 mmol) in toluene (210 mL) was heated to reflux for 6 h
and cooled to room temperature. The toluene was removed
under reduced pressure. The residue was dissolved in CH2Cl2
and filtered through a pad of silica gel to give 6 (17.3 g, 90%)
as a light yellow solid: Anal. RP-HPLC tR ) 1.95 min (column
A, purity 95%); 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 8.14
(s, 1H), 4.31 (hep, 1H, J ) 7.2 Hz), 4.21 (q, 2H, J ) 7.0 Hz),
1.46 (d, 6H, J ) 7.2 Hz), 1.27 (t, 3H, J ) 7.0 Hz).
Ethyl 5-Isopropyl-4-(3-(methoxycarbamoyl)phenoxy)-
pyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (8). To a solu-
tion of 3-hydroxybenzoic acid (0.69 g, 5.0 mmol) in THF (50
mL) were added EDCI (1.9 g, 10 mmol) and HOBt (1.4 g, 10
mmol) at room temperature. After 5 min, diisopropylethyl-
amine (0.71 g, 5.5 mmol) and methyl hydroxylamine hydro-
chloride (0.63 g, 7.5 mmol) were added to the reaction, and
the resulting mixture was stirred for 6 h. The reaction was
diluted with EtOAc (150 mL) and washed with water (3 × 100
mL), 1 N HCl (1 × 50 mL), and brine (1 × 50 mL). The EtOAc
layer was dried (Na2SO4) and concentrated in vacuo to give
3-hydroxy-N-methoxybenzamide (0.72 g, 86%), which was
sufficiently pure to use in the subsequent step.
The crude 3-hydroxy-N-methoxybenzamide (25 mg, 0.15
mmol) was dissolved in DMF (2 mL) and treated with K2CO3
(21 mg, 0.15 mmol), and the resulting suspension was stirred
at room temperature for 5 min. Ethyl 4-chloro-5-isopropylpyr-
rolo[2,1-f][1,2,4]triazine-6-carboxylate was added to the reac-
tion and the mixture was stirred for 18 h. The resulting solid
was filtered and washed with MeOH. The filtrate was con-
centrated in vacuo to give a yellow oil, which was dissolved in
MeOH and purified by preparative HPLC to afford 8 (18 mg,
60%) as a clear film: Anal. RP-HPLC tR ) 1.89 min (column
A, purity 98%); 1H NMR (400 MHz, CD3OD) δ 8.23 (s, 1H),
7.92 (s, 1H), 7.75 (d, 1 H, J ) 8.1 Hz), 7.69 (s, 1H), 7.60 (t, 1H,
J ) 8.1 Hz), 7.50 (d, 1H, J ) 8.1 Hz), 4.37-4.31 (m, 3H), 3.82
(s, 3H), 1.48 (d, 6H, J ) 7.1 Hz), 1.39 (t, 3H, J ) 7.1 Hz); 13C
NMR (125 MHz, CDCl3) δ 164.2, 163.1, 151.5, 147.0, 137.4,
130.2, 130.0, 125.7, 124.8, 123.6, 121.7, 118.5, 117.0, 114.0,
64.8, 60.6, 25.2, 24.9, 21.5, 14.4; HRMS for C20H22N4O5 (M +
H)+ calcd 399.1624, found 399.1679.
Ethyl 4-(2,4-Difluoro-5-(methoxycarbamoyl)phenyl-
amino)-5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carbox-
ylate (14). Representative Procedure for the Prepara-
tion of a m-Amino-N-methoxybenzamide Intermediate
7 and Subsequent Coupling to Ethyl 4-Chloro-5-isopro-
pylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate. To a solu-
tion of 2,4-difluoro-5-nitrobenzoic acid40 (5.00 g, 24.6 mmol)
in EtOH (250 mL) was added tin chloride dihydrate (27.7 g,
123 mmol). The reaction mixture was heated to 50 °C for 10
min and then cooled to room temperature. EtOH was removed
from the reaction mixture under reduced pressure. Ice water
was added to the resulting residue, which was neutralized with
saturated aqueous NaHCO3 to pH 7 and extracted with EtOAc
(3 × 300 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo to give 5-amino-2,4-difluorobenzoic acid
(3.20 g, 18.5 mmol, 75%), which was used directly in the
subsequent reaction without further purification.
Chemistry. All nonaqueous reactions were carried out
under an argon or nitrogen atmosphere at room temperature,
unless otherwise noted. All commercial reagents and anhy-
drous solvents were purchased from Aldrich and were used
without further purification or distillation, unless otherwise
stated. Analytical thin-layer chromatography (TLC) was per-
formed on EM Science silica gel 60 F254 (0.25 mm). Compounds
were visualized by UV light and/or stained with either p-
anisaldehyde, potassium permanganate, or cerium molybdate
solutions followed by heating. Flash column chromatography
was performed on EM Science silica gel 60 (particle size of
40-63 µm). Analytical high-pressure liquid chromatography
(HPLC) and LC-MS analyses were conducted using Shimadzu
LC-10AS pumps and a SPD-10AV UV-vis detector set at 220
nm with the MS detection performed with a Micromass
Platform LC spectrometer. Analytical HPLC analyses were
performed using one of the following conditions: column A,
phenom-prime S5 C18 4.6 × 30 mm column; solvent A, 10%
MeOH-90% H2O-0.1% TFA; solvent B, 90% MeOH-10%
H2O-0.1% TFA; flow rate ) 4 mL/min; linear gradient time
) 2 min; start %B ) 0, final %B ) 100; or column B,
Phenomenex Su C18 4.6 × 50 mm column; solvent A, 10%
MeOH-90% H2O-0.1% TFA; solvent B, 90% MeOH-10%
H2O-0.1% TFA; flow rate ) 4 mL/min; linear gradient time
) 4 min; start %B ) 0, final %B ) 100. Preparative reverse
phase (RP) HPLC was performed using two Shimadzu LC-8A
pumps, a SPD-10AV UV-vis detector set at 220 nm, and a
YMC C18 ODS-A 5 µm, 20 × 100 mm column (eluting at 20
mL/min with a 12 min gradient of 0-100% B, where solvent
A ) 10% MeOH-90% H2O-0.1% TFA and solvent B ) 90%
MeOH-10% H2O-0.1% TFA). Following preparative HPLC
purification, the fractions containing the desired product were
concentrated under reduced pressure to remove the MeOH,
neutralized with saturated aqueous NaHCO3 to pH 7 and
extracted with ethyl acetate (3 × 5 mL). The organic layers
were dried (Na2SO4) and concentrated in vacuo to afford the
desired compounds.
NMR (1H and 13C) spectra were recorded on JEOL GSX 500
MHz or Bruker ARX 400 MHz spectrometers and calibrated
using an internal reference. High-resolution mass spectra
(HMRS) were recorded on a JEOL SX102 mass spectrometer.
Elemental analyses were performed by Robertson Microlit
Laboratories, and the results obtained are within (0.4% of
the theoretical values.
Diethyl 1-Amino-3-isopropyl-1H-pyrrole-2,4-dicarbox-
ylate (4). To a solution of diethyl 3-isopropyl-1H-pyrrole-2,4-
dicarboxylate26 (3, 3.75 g, 14.8 mmol) in DMF (280 mL) at 0
°C was added NaH (60% dispersion in mineral oil, 0.890 g,
22.2 mmol) and the suspension was stirred for 10 min.
O-(Diphenylphosphinyl)hydroxylamine (6.91 g, 29.6 mmol) was
added to the reaction mixture and stirring was continued for
2 h. The reaction was quenched by the addition of pH 7
phosphate buffer (100 mL) and the mixture was extracted with
EtOAc (4 × 100 mL). The combined EtOAc layers were dried
(Na2SO4) and concentrated in vacuo. The residue was purified
by flash chromatography (SiO2, 20-25% EtOAc/hexanes gradi-
ent elution) to give 4 (3.38 g, 85%) as a white solid: Anal. RP-
1
HPLC tR ) 1.29 min (column A, purity 97%); H NMR (400
MHz, CDCl3) δ 7.49 (s, 1H), 5.56 (s, 2H), 4.37 (q, 2H, J ) 7.1
Hz), 4.32 (q, 2H, J ) 7.1 Hz), 3.99 (hep, 1H, J ) 7.2 Hz), 1.42-
1.35 (m, 12H); MS (ESI) m/z 269 (M + H)+.
EDCI (9.45 g, 49.2 mmol) and HOBt (6.64 g, 49.2 mmol)
were added to a solution of 5-amino-2,4-difluorobenzoic acid
(4.26 g, 24.6 mmol) in THF (500 mL) and stirred at room
temperature for 5 min. Diisopropyl ethylamine (3.30 g, 25.0
mmol) and methyl hydroxylamine hydrochloride (3.10 g, 37.5
Ethyl 5-Isopropyl-4-oxo-3,4-dihydropyrrolo[2,1-f][1,2,4]-
triazine-6-carboxylate (5). A solution of 4 (4.8 g, 18 mmol)
in formamide (18 mL) was heated to 165 °C for 6 h and cooled

4000 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
mmol) were then added to the reaction and the mixture was
stirred for 6 h. The reaction mixture was diluted with EtOAc
(500 mL) and washed with saturated aqueous NaHCO3 (3 ×
200 mL), 1 N HCl (2 × 200 mL), and brine (1 × 200 mL). The
EtOAc layer was dried (Na2SO4) and concentrated in vacuo.
The resulting oil was purified by flash chromatography (SiO2,
0-2% MeOH/CHCl3 gradient elution) to give 5-amino-2,4-
difluoro-N-methoxybenzamide (2.98 g, 14.8 mmol, 60%) as an
off-white solid: Anal. RP-HPLC tR ) 0.97 min (column A,
purity 99%); 1H NMR (400 MHz, CD3OD) δ 7.19 (dd, 1H, J )
7.0 Hz), 6.95 (t, 1H, J ) 10.5 Hz), 3.81 (s, 3H); 13C NMR (125
MHz, CD3OD) δ 163.5, 153.5 (dd, J ) 247, 11 Hz), 152.5 (dd,
J ) 241, 10 Hz), 133.9 (d, J ) 13 Hz), 117.4, 117.0 (d, J ) 15
Hz), 104.6 (dd, J ) 28, 26 Hz), 64.0; HRMS for C8H8F2N2O2
(M - H)- calcd 201.0475, found 201.0474. Anal. (C8H8F2N2O2)
C, H, N.
A solution of 5-amino-N-methoxy-2,4-difluorobenzamide (20
mg, 0.10 mmol) and ethyl 4-chloro-5-isopropylpyrrolo[2,1-f]-
[1,2,4]triazine-6-carboxylate (27 mg, 0.10 mmol) in DMF (2
mL) was stirred for 18 h. The crude reaction mixture was
purified by preparative HPLC to afford 14 (34 mg, 78% yield)
as a white solid: Anal. RP-HPLC tR ) 1.70 min (column A,
purity 98%); 1H NMR (400 MHz, CDCl3) δ 9.42 (bs, 1H), 9.33
(bs, 1H), 8.18 (s, 1H), 7.60 (t, 1H, J ) 10.4 Hz), 7.17 (t, 1H, J
) 10.4 Hz), 4.40 (q, 2H, J ) 7.2 Hz), 4.13 (hep, 1H, J ) 7.2
Hz), 3.98 (s, 3H), 1.66 (d, 6H, J ) 7.2 Hz), 1.50 (t, 3H, J ) 7.2
Hz); HRMS for C20H21F2N5O4 (M + H)+ calcd 434.1962, found
434.1965.
Ethyl 4-(2,4-Difluoro-5-(ethoxycarbamoyl)phenylami-
no)-5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxy-
late (15): colorless oil; Anal. RP-HPLC tR ) 1.80 min (column
A, purity 98%); 1H NMR (500 MHz, CDCl3) δ 9.21 (bs, 1H),
9.08 (bs, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.48 (s, 1H), 7.04 (t,
1H, J ) 10.4 Hz), 4.38-4.31 (m, 2H), 4.19-4.08 (m, 3H), 1.55
(d, 6H, J ) 7.2 Hz), 1.41-1.33 (m, 6H); 13C NMR (125 MHz,
CDCl3) δ 164.4, 160.9, 156.2 (dd, J ) 242, 11 Hz), 155.7 (dd, J
) 242, 11 Hz), 153.4, 147.8, 126.8 (d, J ) 15 Hz), 126.5, 123.5,
123.2, 115.7 (d, J ) 15 Hz), 115.4, 112.4, 104.4 (dd, J ) 32, 26
Hz), 72.7, 60.5, 25.5, 22.7 (2C), 14.3, 13.5; HRMS for
C21H23F2N5O4 (M + H)+ calcd 448.1796, found 448.1782.
Ethyl 4-(2,4-Difluoro-5-(isopropoxycarbamoyl)phenyl-
amino)-5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carbox-
ylate (16): off-white solid; Anal. RP-HPLC tR ) 3.84 min
(column B, purity 98%); 1H NMR (500 MHz, DMSO-d6) δ 8.99
(bs, 1H), 8.12 (s, 1H), 7.85 (s, 1H), 7.49 (s, 1H), 7.38 (t, 1H, J
) 10.5 Hz), 7.13 (t, 1H, J ) 7.3 Hz), 4.83-4.49 (m, 1H), 4.18
(q, 1H, J ) 7.0 Hz), 4.09 (hep, 1H, J ) 7.2 Hz), 1.39-1.33 (m,
9H), 1.16 (d, 6H, J ) 7.2 Hz); 13C NMR (125 MHz, DMSO-d6)
δ 163.6, 160.7, 155.5 (dd, J ) 245, 10 Hz), 154.0 (dd, J ) 240,
10 Hz), 149.0, 141.4, 139.0, 132.3 (d, J ) 15 Hz), 124.2, 118.1,
117.0 (d, J ) 15 Hz), 115.6, 112.0, 104.4 (t, J ) 30 Hz), 76.8,
59.7, 24.2, 22.8, 22.1, 21.1, 20.6, 14.3; HRMS for C22H25F2N5O4
(M + H)+ calcd 462.1953, found 462.1964.
Ethyl 4-(2,4-Difluoro-5-(isobutoxycarbamoyl)phenyl-
amino)-5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carbox-
ylate (17): off-white solid; Anal. RP-HPLC tR ) 3.78 min
(column B, purity 96%); 1H NMR (500 MHz, CDCl3) δ 9.21 (bs,
1H), 9.11 (bs, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.49 (s, 1H), 7.02
(t, 1H, J ) 8.0 Hz), 4.37-4.31 (m, 2H), 4.15 (hep, 1H, J ) 7.2
Hz), 3.87-3.63 (m, 2H), 2.06-2.01 (m, 1H), 1.58-1.52 (m, 6H),
1.33 (t, 3H, J ) 7.0 Hz), 1.06 (d, 6H, J ) 6.9 Hz); 13C NMR
(125 MHz, CDCl3) δ 164.4, 160.7, 156.1 (dd, J ) 247, 12 Hz),
155.5 (dd, J ) 247, 12 Hz), 153.4, 147.8, 127.0, 126.5, 123.7
(d, J ) 13 Hz), 123.2, 115.9 (d, J ) 13 Hz), 115.4, 112.4, 104.3
(t, J ) 28 Hz), 83.6, 60.5, 27.4, 25.5, 22.7 (2C), 19.2 (2C), 14.4;
HRMS for C23H27F2N5O4 (M + H)+ calcd 476.2109, found
476.2099.
Ethyl 5-Isopropyl-4-(3-(methoxycarbamoyl)phenylami-
no)pyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (9): clear
film; Anal. RP-HPLC tR ) 1.88 min (column A, purity 97%);
1H NMR (400 MHz, CD3OD) δ 8.33 (s, 1H), 7.95 (s, 1H), 7.92-
7.88 (m, 1H), 7.81 (s, 1H), 7.73-7.69 (m, 2H), 4.35 (q, 2H, J )
7.0 Hz), 3.89-3.78 (m, 4H), 1.55 (d, 6H, J ) 7.2 Hz), 1.39 (t,
3H, J ) 7.0 Hz); 13C NMR (125 MHz, CDCl3 with 1 drop CD3-
OD) δ 165.6, 163.7, 150.9, 150.4, 141.2, 138.5, 134.0, 130.4,
128.2, 126.7, 123.8, 121.6, 116.8, 110.7, 63.9, 60.9, 26.5, 24.7,
21.8, 14.0; HRMS for C20H23N5O4 (M + H)+ calcd 398.1828,
found 398.1821.
Ethyl 4-(5-((Cyclopropylmethoxy)carbamoyl)-2,4-di-
fluorophenylamino)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazine-6-carboxylate (18): off-white solid; Anal. RP-HPLC
tR ) 3.81 min (column B, purity 98%); 1H NMR (500 MHz,
CDCl3) δ 9.19-9.16 (m, 2H), 8.07 (s, 1H), 8.01 (s, 1H), 7.49 (s,
1H), 7.02 (t, 1H, J ) 10.0 Hz), 4.34 (q, 2H, J ) 7.1 Hz), 4.19-
4.11 (m, 1H), 3.90 (d, 2H, J ) 7.1 Hz), 1.55 (d, 6H, J ) 7.7
Hz), 1.40 (t, 3H, J ) 7.1 Hz), 1.29-1.18 (m, 1H), 0.68-0.61
(m, 2H), 0.39-0.33 (m, 2H); 13C NMR (125 MHz, CDCl3) δ
164.4, 160.6, 156.4 (dd, J ) 250, 14 Hz), 155.1 (dd, J ) 250,
14 Hz), 153.4, 147.8, 127.0, 126.5, 123.5 (d, J ) 11 Hz), 123.1,
115.4 (d, J ) 17 Hz), 115.1, 112.2, 104.1 (t, J ) 28 Hz), 81.5,
60.4, 25.4, 22.7 (2C), 14.3, 9.1, 3.1 (2C); HRMS for C23H25F2N5O4
(M + H)+ calcd 474.1953, found 474.1956.
4-(2,4-Difluoro-5-(methoxycarbamoyl)phenylamino)-5-
isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic Acid
(19). A mixture of compound 14 (6.0 g, 14 mmol) and 1 N
NaOH (140 mL) in THF (140 mL) was heated to 60 °C for 27
h and then cooled to room temperature. The reaction mixture
was acidified to pH 4 with 1 N HCl, and the volatiles were
removed in vacuo. The precipitate was filtered, washed with
diethyl ether, and dried under high vacuum overnight to give
19 (5.5 g, 98%) as an off-white solid: Anal. RP-HPLC tR ) 1.33
min (column A, purity 95%); 1H NMR (400 MHz, DMSO-d6) δ
12.28 (bs, 1H), 11.57 (s, 1H), 10.54 (s, 1H), 7.88 (s, 1H), 7.51-
7.47 (m, 1H), 7.45 (t, 1H, J ) 10.5 Hz), 7.22-7.18 (m, 1H),
4.46 (hep, 1H, J ) 7.1 Hz), 3.70 (s, 3H), 1.34 (d, 6H, J ) 7.1
Hz); MS (ESI) m/z 406 (M + H)+.
Ethyl 5-Isopropyl-4-(3-(methoxycarbamoyl)-4-meth-
ylphenylamino)pyrrolo[2,1-f][1,2,4]triazine-6-carboxy-
late (10): clear film; Anal. RP-HPLC tR ) 1.68 min (column
A, purity 98%); 1H NMR (400 MHz, CD3OD) δ 8.08 (s, 1H),
7.69 (s, 1H), 7.43-7.38 (m, 3H), 4.34 (q, 2H, J ) 7.0 Hz), 3.98
(hep, 1H, J ) 7.1 Hz), 3.84 (s, 3H), 2.45 (s, 3H), 1.51 (d, 6H, J
) 7.1 Hz), 1.38 (t, 3H, J ) 7.0 Hz); HRMS for C21H25N5O4 (M
+ H)+ calcd 412.1947, found 412.1984.
Ethyl 4-(4-Bromo-3-(methoxycarbamoyl)phenylamino)-
5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (11):
off-white solid; Anal. RP-HPLC tR ) 1.76 min (column A, purity
97%); 1H NMR (400 MHz, CD3OD) δ 7.95 (s, 1H), 7.68 (d, 1H,
J ) 8.6 Hz), 7.61 (s, 1H), 7.41 (s, 1H), 7.33 (d, 1H, J ) 6.9
Hz), 4.31 (q, 2H, J ) 7.1 Hz), 4.12 (hep, 1H, J ) 7.0 Hz), 3.85
(s, 3H), 1.47 (d, 6H, J ) 7.0 Hz), 1.40 (t, 3H, J ) 7.1 Hz); HRMS
for C20H22BrN5O4 (M + H)+ calcd 476.0933, found 476.0930.
Ethyl 4-(4-Fluoro-3-(methoxycarbamoyl)phenylamino)-
5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (12):
off-white solid; Anal. RP-HPLC tR ) 1.80 min (column A, purity
1
97%); H NMR (400 MHz, CDCl3) δ 9.35 (bs, 1H), 8.14-8.12
(m, 2H), 8.06 (s, 1H), 7.95 (s, 1H), 7.43 (bs, 1H), 7.21 (t, 1H, J
) 10.1 Hz), 4.35 (q, 2H, J ) 7.1 Hz), 4.13 (hep, 1H, J ) 7.2
Hz), 3.92 (s, 3H), 1.57 (d, 6H, J ) 7.2 Hz), 1.40 (t, 3H, J ) 7.1
Hz); HRMS for C20H22FN5O4 (M + H)+ calcd 416.1734, found
416.1737.
Ethyl 4-(2-Fluoro-5-(methoxycarbamoyl)phenylamino)-
5-isopropylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (13):
off-white solid; Anal. RP-HPLC tR ) 1.77 min (column A, purity
96%); 1H NMR (400 MHz, CDCl3) δ 9.09 (bs, 1H), 8.81 (bs,
1H), 8.07 (s, 1H), 8.04 (s, 1H), 7.75-7.73 (m, 1H), 7.60-7.56
(m, 1H), 7.25 (t, 1H, J ) 10.5 Hz), 4.35 (q, 2H, J ) 7.1 Hz),
4.17-4.11 (m, 1H), 3.93 (s, 3H), 1.55 (d, 6H, J ) 7.3 Hz), 1.40
(t, 3H, J ) 7.1 Hz); HRMS for C20H22FN5O4 (M + H)+ calcd
416.1734, found 416.1737.
5-(6-(Azidocarbonyl)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazin-4-ylamino)-2,4-difluoro-N-methoxybenzamide (20).
A solution of compound 19 (0.41 g, 0.94 mmol) in anhydrous
1,4-dioxane (10 mL) was treated with triethylamine (0.20 mL,
1.4 mmol) and diphenylphosphoryl azide (0.39 g, 1.4 mmol).
The reaction mixture was heated to 40 °C for 1.5 h and then
cooled to room temperature and concentrated under reduced

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4001
pressure. The residue was passed through a silica gel pad
eluting with 25% EtOAc/hexanes to give 20 (0.42 g, 99%) as a
pale yellow oil, which was used directly in the next step
without further purification: Anal. RP-HPLC tR ) 1.86 min
(column A, purity 90%); 1H NMR (400 MHz, CDCl3) δ 9.40 (bs,
1H), 9.10 (bs, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.78 (s, 1H,), 7.54
(t, 1H, J ) 10.3 Hz), 4.15 (hep, 1H, J ) 7.2 Hz), 3.91 (s, 3H),
1.60 (d, 6H, J ) 7.2 Hz); MS (ESI) m/z 406 (M + H)+.
g, 16 mmol) in THF/water (100/10 mL) was treated with 1 N
NaOH (82 mL) and the resulting mixture was heated to 60 °C
for 8 h, cooled to room temperature, and concentrated under
reduced pressure to remove the THF. The remaining solution
was acidified with 1 N HCl to pH 3. The precipitate was
filtered, washed with water, and dried under high vacuum
overnight to give 27 (3.4 g, 95%) as a white solid, which was
used directly in the next step without further purification:
Anal. RP-HPLC tR ) 2.30 min (column B, purity 98%); 1H NMR
(500 MHz, DMSO-d6) δ 12.40 (bs, 1H), 11.63 (bs, 1H), 7.86 (s,
1H), 7.83 (s, 1H), 4.21 (hep, 1H, J ) 7.1 Hz), 1.31 (d, 6H, J )
7.1 Hz); 13C NMR (125 MHz, DMSO-d6) δ 165.0, 154.6, 139.8,
134.4, 124.2, 117.1, 113.6, 23,7, 21.4 (2C); HRMS for C10H11N3O3
(M - H)- calcd 220.0800, found 220.0728.
2,4-Difluoro-5-(5-isopropyl-6-(5-methyloxazol-2-yl)-pyr-
rolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxybenza-
mide (29). A mixture of acid 27 (0.44 g, 2.0 mmol), 1-ami-
noacetone HCl salt41 (0.27 g, 2.5 mmol), BOP reagent (1.1 g,
2.5 mmol), and triethylamine (0.68 mL, 4.9 mmol) in DMF (5
mL) was stirred at 50 °C for 2 h. The reaction mixture was
partitioned between ethyl acetate (10 mL) and water (5 mL).
The ethyl acetate layer was washed with brine, dried (MgSO4),
and concentrated under reduced pressure. The residue was
then purified by flash chromatography (SiO2; 100% ethyl
acetate) to afford 4-hydroxy-5-isopropyl-N-(2-oxopropyl)pyr-
rolo[2,1-f][1,2,4]triazine-6-carboxamide (0.34 g, 61%) as a pale
yellow solid, which was used directly in the next step.
Ethyl 4-(2,4-Difluoro-5-(methoxycarbamoyl)phenyl-
amino)-5-isopropylpyrrolo[2,1-f][1,2,4]triazin-6-ylcar-
bamate (21). Representative Procedure for the Prepara-
tion of C6-Carbamate Derivatives. A mixture of compound
20 (50 mg, 0.12 mmol) and EtOH (53 mg, 1.2 mmol) in DMF
(1.2 mL) was heated to 85 °C for 2 h. The reaction mixture
was cooled to room temperature, concentrated under reduced
pressure, and purified by flash chromatography (SiO2, 25-
40% EtOAc/hexanes gradient elution) to give 21 (32 mg, 62%)
as an amorphous solid: Anal. RP-HPLC tR ) 3.09 min (column
1
B, purity 98%); H NMR (500 MHz, CD3OD) δ 8.01-7.97 (m,
1H), 7.62-7.58 (m, 2H), 7.02 (t, 1H, J ) 10.1 Hz), 4.10 (q, 2H,
J ) 7.0 Hz), 3.72 (s, 3H), 3.58 (hep, 1H, J ) 7.2 Hz), 1.35 (d,
6H, J ) 7.2 Hz), 1.20 (q, 3H, J ) 7.0 Hz); HRMS for
C20H22F2N6O4 (M + H)+ calcd 449.1749, found 449.1753.
Benzyl 4-(2,4-Difluoro-5-(methoxycarbamoyl)phenyl-
amino)-5-isopropylpyrrolo[2,1-f][1,2,4]triazin-6-ylcar-
bamate (22): amorphous solid; Anal. RP-HPLC tR ) 1.70 min
1
(column A, purity 97%); H NMR (400 MHz, CD3OD) δ 7.98
(m, 1H), 7.71 (s, 1H), 7.40-7.18 (m, 6H), 7.12 (t, 1H, J ) 10.0
Hz), 5.19 (s, 2H), 3.81 (s, 3H), 3.65 (hep, 1H, J ) 7.2 Hz), 1.42
(d, 6H, J ) 7.2 Hz); MS (ESI) m/z 511 (M + H)+.
The above carboxamide (55 mg, 0.21 mmol) was stirred with
POCl3 (2 mL) at 120 °C for 6 h. The excess POCl3 was removed
in vacuo and the residue was partitioned between dichlo-
romethane (10 mL) and saturated aqueous NaHCO3 solution
(10 mL). The organic layer was dried (MgSO4) and concen-
trated under reduced pressure. The residue was purified by
flash chromatography (SiO2, 50% ethyl acetate/hexane) to
afford chloroimidate 28 (31 mg, 0.11 mmol, 55%) as a pale
yellow oil: Anal. RP-HPLC tR ) 1.87 min (column A, purity
3-(Piperidin-1-yl)propyl 4-(2,4-difluoro-5-(methoxycar-
bamoyl)phenylamino)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazin-6-ylcarbamate, Hydrochloride Salt (23): amor-
phous solid; Anal. RP-HPLC tR ) 2.28 min (column B, purity
98%); 1H NMR (500 MHz, CD3OD) δ 8.05-7.99 (m, 1H), 7.62-
7.58 (m, 2H), 7.22 (t, 1H, J ) 10.1 Hz), 4.28-4.23 (m, 2H),
3.85 (s, 3H), 3.69-3.62 (m, 2H), 3.26-3.23 (m, 2H), 2.98-2.91
(m, 2H), 2.20-2.11 (m, 2H), 2.10-1.91 (m, 2H), 1.89-1.74 (m,
4H), 1.59-1.50 (m, 1H), 1.47 (d, 6H, J ) 7.1 Hz); 13C NMR
(125 MHz, CDCl3) δ 163.7, 162.2, 161.5, 158.0, 156.4 (dd, J )
251, 12 Hz), 155.1 (dd, J ) 251, 12 Hz), 151.8, 145.5, 127.0 (d,
J ) 15 Hz), 124.4, 123.5, 115.1 (d, J ) 15 Hz), 113.8, 104.4 (t,
J ) 28 Hz), 64.8, 61.9, 54.4, 53.4, 37.0, 34.9, 31.7, 25.7, 23.8,
23.3, 22.7, 22.0; HRMS for C26H33F2N7O4 (M + H)+ calcd
546.2640, found 546.2616.
1
98%); H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.12 (s, 1H),
6.90 (s, 1H), 4.33 (hep, 1H, J ) 7.2 Hz), 2.41 (s, 3H), 1.46 (d,
6H, J ) 7.2 Hz).
A solution of 28 (31 mg, 0.11 mmol) and 5-amino-2,4-
difluoro-N-methoxybenzamide (24 mg, 0.12 mmol) in MeCN
(3 mL) was stirred at room temperature for 18 h. The solvent
was removed under reduced pressure and the residue was
purified by preparative HPLC to afford 29 (42 mg, 86%) as a
white solid: Anal. RP-HPLC tR ) 1.71 min (column A, purity
1
3-(Methylsulfonyl)propyl 4-(2,4-difluoro-5-(methoxy-
carbamoyl)phenylamino)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazin-6-ylcarbamate (24): amorphous solid; Anal. RP-
97%); H NMR (500 MHz, CD3OD) δ 7.80 (s, 1H), 7.71-7.66
(m, 1H), 7.49 (s, 1H), 7.14 (t, 1H, J ) 10.1 Hz), 6.86 (s, 1H),
4.04 (hep, 1H, J ) 7.2 Hz), 3.24 (s, 3H), 2.33 (s, 3H), 1.35 (d,
6H, J ) 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 160.9, 157.1,
156.5 (dd, J ) 248, 10 Hz), 155.5 (dd, J ) 250, 10 Hz), 152.8,
148.1, 147.1, 146.8, 126.8, 123.8, 122.9, 119.6, 115.0 (d, J )
12 Hz), 113.3, 112.1, 105.5 (dd, J ) 29, 27 Hz), 64.8, 25.4, 23.0
(2C), 10.9; HRMS for C21H20F2N6O3 (M + H)+ calcd 443.1643,
found 443.1648.
1
HPLC tR ) 2.51 min (column B, purity 98%); H NMR (500
MHz, DMSO-d6) δ 8.78 (s, 1H), 7.68-7.55 (m, 3H), 7.50-7.41
(m, 1H), 4.18-4.10 (m, 2H), 3.69 (s, 3H), 3.67-3.60 (m, 2H),
3.26-3.19 (m, 2H), 3.00 (s, 3H), 2.09-1.98 (m, 2H), 1.32 (d,
6H, J ) 7.2 Hz); HRMS for C22H26F2N6O6S (M + H)+ calcd
541.1637, found 541.1635.
(Tetrahydrofuran-2-yl)methyl 4-(2,4-difluoro-5-(meth-
oxycarbamoyl)phenylamino)-5-isopropylpyrrolo[2,1-f]-
[1,2,4]triazin-6-ylcarbamate (25): amorphous solid; Anal.
RP-HPLC tR ) 2.91 min (column B, purity 98%); 1H NMR (500
MHz, DMSO-d6) δ 11.8 (bs, 1H), 8.84 (bs, 1H), 7.68-7.40 (m,
4H), 4.18-3.97 (m, 2H), 3.71 (s, 3H), 3.71-3.57 (m, 3H), 1.97-
1.81 (m, 3H), 1.67-1.60 (m, 1H), 1.32 (d, 6H, J ) 6.9 Hz);
HRMS for C23H26F2N6O5 (M + H)+ calcd 505.1936, found
505.1931.
Methylpiperidin-4-yl 4-(2,4-difluoro-5-(methoxycar-
bamoyl)phenylamino)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazin-6-ylcarbamate, Hydrochloride Salt (26): amor-
phous solid; Anal. RP-HPLC tR ) 1.03 min (column A, purity
94%); 1H NMR (500 MHz, CD3OD) δ 8.38 (bs, 1H), 7.83 (s,
1H), 7.76 (s, 1H), 7.39-7.11 (m, 4H), 4.98-4.89 (m, 2H), 3.83
(s, 3H), 3.63-3.54 (m, 1H), 3.29-3.18 (m, 1H), 3.10-2.99 (m,
2H), 2.72 (s, 3H), 2.30-2.23 (m, 2H), 2.10-2.01 (m, 2H), 1.49
(bs, 6H); MS (ESI) m/z 518 (M + H)+.
2,4-Difluoro-5-(5-isopropyl-6-(3-methyl-1,2,4-oxadiazol-
5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxy-
benzamide (31). To a solution of acid 27 (0.20 g, 0.90 mmol),
N-hydroxylbenzotriazole (24 mg, 0.018 mmol), and diisopro-
pylethylamine (0.58 g, 4.5 mmol) in DMF (9 mL) was added
benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate (0.29 g, 0.90 mmol) at room temperature. N-Hydroxyl-
acetamidine was added to the reaction and the resulting
mixture was stirred for 14 h. The reaction mixture was
quenched with water (7 mL) and extracted with EtOAc (3 ×
10 mL). The EtOAc layers were washed with 10% aqueous LiCl
(2 × 5 mL), dried (Na2SO4), and concentrated under reduced
pressure to give the desired intermediate, which was used
directly in the next step without further purification: MS (ESI)
m/z 278 (M + H)+.
The above intermediate was dissolved in DMF (9 mL) and
heated to 140 °C for 3 h. The reaction mixture was cooled to
room temperature, diluted with EtOAc (30 mL), and washed
with 10% aqueous LiCl (3 × 50 mL). The organics were dried
(Na2SO4), concentrated in vacuo, and triturated with 10%
5-Isopropyl-4-oxo-3,4-dihydropyrrolo[2,1-f][1,2,4]-
triazine-6-carboxylic Acid (27). A solution of ester 5 (4.0

4002 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
MeOH in Et2O to give 30 (0.13 g, 0.51 mmol, 57%) as a white
solid: Anal. RP-HPLC tR ) 1.59 min (column A, purity 98%);
1H NMR (400 MHz, DMSO-d6) δ 11.79 (bs, 1 H), 8.25 (s, 1H),
7.92 (s, 1H), 4.18 (hep, 1H, J ) 7.2 Hz), 2.50 (s, 3H), 1.37 (d,
6H, J ) 7.2 Hz); MS (ESI) m/z 260 (M + H)+.
Compound 33 (10 mg, 0.039 mmol) was dissolved in POCl3
(1 mL) and the resulting mixture was heated at 120 °C for 5
h. The excess POCl3 was removed under reduced pressure and
the residue was coevaporated with toluene. The residue was
then dissolved in MeCN and treated with 5-amino-2,4-difluoro-
N-methoxybenzamide (8.0 mg, 0.040 mmol) and the mixture
was stirred at room temperature for 14 h. The solvent was
removed under reduced pressure and the residue was purified
by preparative HPLC to afford 34 (5.2 mg, 30%) as an off-
white solid: Anal. RP-HPLC tR ) 1.49 min (column A, purity
Intermediate 30 (65 mg, 0.25 mmol) was treated with POCl3
(2 mL) at 120 °C for 6 h. The excess POCl3 was removed under
reduced pressure and the residue was coevaporated with
toluene. The residue was then dissolved in MeCN (3 mL) and
treated with 5-amino-2,4-difluoro-N-methoxybenzamide (51
mg, 0.25 mmol). The reaction mixture was stirred at room
temperature for 18 h. The solvent was removed under reduced
pressure and the residue was purified by preparative HPLC
to afford 31 (73 mg, 68%) as an off-white solid: Anal. RP-HPLC
tR ) 3.11 min (column B, purity 98%); 1H NMR (500 MHz,
CD3OD) δ 8.24 (s, 1H), 7.86-7.81 (m, 1H), 7.68 (s, 1H), 7.32
(t, 1H, J ) 10.5 Hz), 4.11-4.07 (m, 1H), 3.78 (s, 3H), 2.40 (s,
3H), 1.47 (d, 6H, J ) 6.6 Hz); 13C NMR (125 MHz, CD3OD) δ
171.8, 166.7, 159.8, 156.6 (dd, J ) 250, 10 Hz), 154.5 (dd, J )
245, 10 Hz), 140.9, 138.7, 132.3, 131.5, 124.5, 122.1, 117.8,
115.3 (d, J ) 15 Hz), 113.7, 105.2 (t, J ) 28 Hz), 63.0, 24.4,
20.8 (2C), 11.0; HRMS for C20H19F2N7O3 (M + H)+ calcd
444.1596, found 444.1590.
5-Isopropyl-4-oxo-3,4-dihydropyrrolo[2,1-f][1,2,4]-
triazine-6-carbonitrile (32). A mixture of acid 27 (0.39 g,
1.8 mmol), concentrated NH4OH (2 mL), EDCI (0.44 g, 2.3
mmol), and HOBt (0.26 g, 1.9 mmol) in DMF (8 mL) was
stirred at room temperature for 4 h. The reaction mixture was
diluted with water (10 mL) and extracted with EtOAc (3 × 20
mL). The EtOAc layer was dried (Na2SO4), concentrated under
reduced pressure, and triturated with diethyl ether to give
5-isopropyl-4-oxo-3,4-dihydropyrrolo[2,1-f][1,2,4]triazine-6-car-
boxamide (0.36 g, 92%) as an off-white solid: Anal. RP-HPLC
tR ) 1.60 min (column A, purity 90%); MS (ESI) m/z 221 (M +
H)+.
The above amide (60 mg, 0.27 mmol) was dissolved in POCl3
(1 mL), heated to 60 °C under N2 for 1 h, and then cooled to
room temperature. The POCl3 was removed under reduced
pressure and coevaporated with toluene. The residue was
dissolved in EtOAc (5 mL) and washed with saturated aqueous
NaHCO3. The organics were dried (Na2SO4), concentrated in
vacuo, and triturated with 10% MeOH in diethyl ether to
afford compound 32 (50 mg, 88%) as an off-white solid: Anal.
RP-HPLC tR ) 1.45 min (column A, purity 90%); 1H NMR (400
MHz, DMSO-d6) δ 7.56 (s, 1H), 7.23 (s, 1H), 4.21 (hep, 1H, J
) 7.2 Hz), 1.31 (d, 6H, J ) 7.2 Hz); MS (ESI) m/z 201 (M -
H)-.
2,4-Difluoro-5-(5-isopropyl-6-(5-methyl-1,2,4-oxadiazol-
3-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxy-
benzamide (34). A mixture of compound 32 (70 mg, 0.35
mmol), hydroxylamine hydrochloride (37 mg, 1.0 mmol), and
K2CO3 (0.23 g, 1.7 mmol) in EtOH (4 mL) was heated to reflux
for 28 h and then cooled to room temperature. Water (4 mL)
was added to the reaction and the mixture was extracted with
EtOAc (3 × 10 mL). The combined EtOAc layers were washed
with brine (1 × 10 mL), dried (Na2SO4), and concentrated
under reduced pressure to give N′-hydroxy-5-isopropyl-4-oxo-
3,4-dihydropyrrolo[2,1-f][1,2,4]triazine-6-carboxamidine, which
was used directly in the next step without further purifica-
tion: Anal. RP-HPLC tR ) 1.38 min (column A, purity 90%);
MS (ESI) m/z 335 (M + H)+.
1
98%); H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 7.88 (s,
1H), 7.58 (s, 1H), 7.48 (t, 1H, J ) 10.4 Hz), 7.31 (m, 1H), 5.5
(bs, 1 H), 4.25 (hep, 1H, J ) 7.2 Hz), 3.71 (s, 3H), 2.65 (s, 3H),
1.39 (d, 6H, J ) 7.2 Hz); 13C NMR (125 MHz, CD3OD) δ 178.5,
163.4, 160.4, 155.0 (dd, J ) 250, 10 Hz), 154.5 (dd, J ) 245,
10 Hz), 140.9, 138.7, 132.3, 131.5, 123.0 (d, J ) 10 Hz), 122.1,
117.8, 115.3 (d, J ) 15 Hz), 113.7, 105.5 (t, J ) 30 Hz), 63.0,
26.4, 22.3 (2C), 14.5; HRMS for C20H19F2N7O3 (M + H)+ calcd
444.1596, found 444.1580.
5-Isopropyl-4-oxo-3,4-dihydropyrrolo[2,1-f][1,2,4]-
triazine-6-carbohydrazide (35). A mixture of ester 5 (20.0
g, 80.3 mmol), hydrazine (80 mL), and EtOH (20 mL) was
heated to 90 °C for 4 h, cooled to room temperature, and con-
centrated under reduced pressure. The residue was triturated
with 10% MeOH/Et2O to give 35 (17.9 g, 95%) as an off-white
solid: Anal. RP-HPLC tR ) 0.72 min (column A, purity 95%);
1H NMR (400 MHz, DMSO-d6) δ 9.30 (bs, 1H), 7.78 (s, 1H),
7.76 (s, 1H), 4.36-4.11 (bs, 3H), 4.08 (hep, 1H, J ) 7.2 Hz),
1.30 (d, 6H, J ) 7.2 Hz); MS (ESI) m/z 236 (M + H)+.
2,4-Difluoro-5-(5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-
2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxy-
benzamide (37). Representative Procedure for the Prepa-
ration of Chloroimidate 36 and Subsequent Coupling to
5-Amino-2,4-difluoro-N-methoxybenzamide. A mixture of
hydrazide 35 (0.10 g, 0.43 mmol) and the corresponding
carboxylic acid (acetic acid, 0.2 mL) in POCl3 (2 mL) was
heated to 100 °C for 1 h and then at 120 °C for 3 h under N2.
The reaction mixture was cooled to room temperature. The
POCl3 was removed under reduced pressure and coevaporated
with toluene. The residue was dissolved in EtOAc (5 mL) and
washed with cold saturated aqueous NaHCO3. The EtOAc
layer was dried (Na2SO4) and passed through a short silica
gel pad to give 4-chloro-5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-
2-yl)-pyrrolo[2,1-f][1,2,4]triazine (36), which was used directly
in the next step without further purification.
4-Chloro-5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)pyrrolo-
[2,1-f][1,2,4]triazine (36, 33 mg, 0.11 mmol) was treated with
5-amino-2,4-difluoro-N-methoxybenzamide (24 mg, 0.12 mmol)
in DMF (3 mL) and the mixture was stirred at 50 °C for 6 h.
The solvent was removed in vacuo and the residue was purified
by preparative HPLC to afford 37 (30 mg, 60%) as a white
solid: Anal. RP-HPLC tR ) 1.58 min (column A, purity 98%);
1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 7.96-7.92 (m, 1H),
7.78 (s, 1H), 7.41 (t, 1H, J ) 10.0 Hz), 4.08 (hep, 1H, J ) 7.1
Hz), 3.85 (s, 3H), 2.65 (s, 3H), 1.54 (d, 6H, J ) 7.1 Hz); HRMS
for C20H19F2N7O3 (M + H)+ calcd 444.1596, found 444.1599.
5-(6-(5-Ethyl-1,3,4-oxadiazol-2-yl)-5-isopropylpyrrolo-
[2,1-f][1,2,4]triazin-4-ylamino)-2,4-difluoro-N-methoxy-
benzamide (39): off-white solid; Anal. RP-HPLC tR ) 1.69
1
min (column A, purity 96%); H NMR (400 MHz, CD3OD) δ
7.98 (s, 1H), 7.76-7.73 (m, 1H), 7.58 (s, 1H), 7.25 (t, 1H, J )
10.1 Hz), 4.24 (hep, 1H, J ) 7.1 Hz), 3.85 (s, 3H), 3.00 (q, 2H,
J ) 7.5 Hz), 1.49 (d, 6H, J ) 7.1 Hz), 1.44 (t, 3H, J ) 7.5 Hz);
HRMS for C21H21F2N7O3 (M + H)+ calcd 458.1698, found
458.1699.
The above amidine was dissolved in pyridine (2 mL) and
treated with acetyl chloride (0.5 mL). The reaction mixture
was heated to reflux for 7 h, cooled to room temperature,
diluted with water (4 mL), and extracted with EtOAc (3 × 10
mL). The organic layers were washed with 1 N NaOH (1 × 5
mL) and 1 N HCl (2 × 5 mL), dried (Na2SO4), concentrated in
vacuo, and purified by flash chromatography (SiO2, 5-10%
MeOH/CH2Cl2 gradient elution) to give 33 (10 mg, 0.039 mmol,
11%) as an off-white solid: Anal. RP-HPLC tR ) 1.25 min
(column A, purity 98%); 1H NMR (400 MHz, DMSO-d6) δ 8.20
(s, 1H), 7.91 (s, 1H), 4.11 (hep, 1H, J ) 7.2 Hz), 2.47 (s, 3H),
1.37 (d, 6H, J ) 7.2 Hz); MS (ESI) m/z 260 (M + H)+.
2,4-Difluoro-5-(5-isopropyl-6-(5-isopropyl-1,3,4-oxadia-
zol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-meth-
oxybenzamide (40): off-white solid; Anal. RP-HPLC tR
)
1.76 min (column A, purity 98%); 1H NMR (400 MHz, CDCl3)
δ 9.26-9.21 (m, 2H), 8.07 (s, 1H), 8.06 (s, 1H), 7.53-7.51 (m,
1H), 7.06 (t, 1H, J ) 10.3 Hz), 4.26-4.22 (m, 1H), 3.94 (s, 3H),
3.32-3.25 (m, 1H), 1.57 (d, 6H, J ) 7.3 Hz), 1.46 (d, 6H, J )
7.1 Hz); HRMS for C22H23F2N7O3 (M + H)+ calcd 472.1888,
found 472.1890.

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4003
2,4-Difluoro-5-(6-(5-isobutyl-1,3,4-oxadiazol-2-yl)-5-iso-
propylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxy-
benzamide (41): off-white solid; Anal. RP-HPLC tR ) 1.87
7.72-7.48 (m, 2H), 7.22 (t, 1H, J ) 10.3 Hz), 4.82 (s, 2H), 4.37-
4.28 (m, 1H), 3.82 (s, 3H), 3.31 (s, 3H), 3.11 (s, 3H), 1.51 (d,
6H, J ) 7.1 Hz); MS (ESI) m/z 487 (M + H)+.
1
min (column A, purity 95%); H NMR (400 MHz, CD3OD) δ
2,4-Difluoro-5-(5-isopropyl-6-(5-(methylsulfonylmethyl)-
1,3,4-oxadiazol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-
N-methoxybenzamide (49): white solid; Anal. RP-HPLC tR
) 2.75 min (column B, purity 96%); 1H NMR (400 MHz, CD3-
OD) δ 8.03 (s, 1H), 7.73-7.66 (m, 1H), 7.52 (s, 1H), 7.16 (t,
1H, J ) 10.0 Hz), 4.83 (s, 2H), 3.93 (hep, 1H, J ) 7.0 Hz),
3.67 (s, 3H), 3.04 (s, 3H), 1.34 (d, 6H, J ) 7.0 Hz); HRMS for
C21H21F2N7O5S (M + H)+ calcd 522.1371, found 522.1369.
2,4-Difluoro-5-(5-isopropyl-6-(1,3,4-oxadiazol-2-yl)pyr-
rolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxybenza-
mide (38). A solution of hydrazide 35 (48 mg, 0.20 mmol) and
triethyl orthoformate (0.2 mL) in EtOH (2 mL) was heated to
120 °C for 7 h. The reaction mixture was cooled to room
temperature, and the volatiles were removed under reduced
pressure. The residue was triturated with Et2O to give
5-isopropyl-6-(1,3,4-oxadiazol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-
4(3H)-one (10 mg, 0.041 mmol, 20%), which was used directly
in the next step without further purification: Anal. RP-HPLC
tR 1.23 min (column A, purity 98%); 1H NMR (400 MHz,
DMSO-d6) δ 11.8 (bs, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.92 (s,
1H), 4.18 (hep, 1H, J ) 7.2 Hz), 2.50 (s, 3H), 1.37 (d, 6H, J )
7.2 Hz); MS (ESI) m/z 246 (M + H)+.
The 5-isopropyl-6-(1,3,4-oxadiazol-2-yl)pyrrolo[2,1-f][1,2,4]-
triazin-4(3H)-one (10 mg, 0.041 mmol) was dissolved in POCl3
(1 mL) and the reaction mixture was stirred at 120 °C for 5 h.
The excess POCl3 was removed under reduced pressure and
the residue was coevaporated with toluene. The residue was
dissolved in MeCN and treated with 5-amino-2,4-difluoro-N-
methoxybenzamide (8.3 mg, 0.041 mmol). The reaction mixture
was stirred at room temperature for 19 h, the solvent was
removed under reduced pressure, and the residue was purified
by preparative HPLC to afford 38 (9.8 mg, 56%) as a white
solid: Anal. RP-HPLC tR ) 1.43 min (column A, purity 98%);
1H NMR (400 MHz, CD3OD) δ 9.00 (s, 1H), 8.01 (s, 1H), 7.71-
7.67 (m, 1H), 7.57 (s, 1H), 7.25 (t, 1H, J ) 10.1 Hz), 4.33-
4.27 (m, 1H), 3.85 (s, 3H), 1.50 (d, 6H, J ) 7.1 Hz); HRMS for
C19H17F2N7O3 (M + H)+ calcd 430.1394, found 430.1399.
5-(6-(5-(Dimethylamino)-1,3,4-oxadiazol-2-yl)-5-isopro-
pylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-2,4-difluoro-N-
methoxybenzamide (47). A solution of hydrazide 35 (92 mg,
0.39 mmol) and phosgene iminium chloride (76 mg, 0.47 mmol)
in MeCN was heated to 80 °C for 6 h. The reaction mixture
was cooled to room temperature and diluted with water (4 mL).
The precipitate that formed after 5 min of stirring was filtered,
washed with water, and dried overnight to give 6-(5-(dimethyl-
amino)-1,3,4-oxadiazol-2-yl)-5-isopropylpyrrolo[2,1-f][1,2,4]-
triazin-4(3H)-one (91 mg, 0.32 mmol, 82%) as a colorless oil:
Anal. RP-HPLC tR ) 1.35 min (column A, purity 97%); 1H NMR
(400 MHz, CD3OD) δ 7.79 (s, 1H), 7.48 (s, 1H), 3.92 (hep, 1H,
J ) 7.2 Hz), 3.07 (s, 3H), 3.05 (s, 3H), 1.27 (d, 6H, J ) 7.2
Hz); MS (ESI) m/z 289 (M + H)+.
7.95 (s, 1H), 7.74-7.72 (m, 1H), 7.57 (s, 1H), 7.24 (t, 1H, J )
10.1 Hz), 4.26-4.22 (m, 1H), 3.84 (s, 3H), 2.86 (d, 2H, J ) 7.1
Hz), 2.28-2.21 (m, 1H), 1.49 (d, 6H, J ) 7.1 Hz), 1.08 (d, 6H,
J ) 6.7 Hz); HRMS for C23H25F2N7O3 (M + H)+ calcd 486.1202,
found 486.1204.
5-(6-(5-Cyclopropyl-1,3,4-oxadiazol-2-yl)-5-isopropyl-
pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-2,4-difluoro-N-
methoxybenzamide (42): off-white solid; Anal. RP-HPLC tR
) 3.57 min (column B, purity 98%); 1H NMR (500 MHz, CDCl3)
δ 9.27-9.22 (m, 2H), 8.03-8.01 (m, 2H), 7.58-7.50 (m, 1H),
7.05 (t, 1H, J ) 10.1 Hz), 4.28-4.21 (m, 1H), 3.93 (s, 3H), 2.26-
2.22 (m, 1H), 1.55 (d, 6H, J ) 7.1 Hz), 1.24-1.18 (m, 4H); 13
C
NMR (125 MHz, CDCl3) δ 167.8, 160.8, 160.5, 156.2 (dd, J )
250, 14 Hz), 155.7 (dd, J ) 254, 14 Hz), 152.9, 147.5, 127.0,
123.7 (d, J ) 41 Hz), 119.9 (2C), 115.6 (d, J ) 15 Hz), 112.8,
109.0, 104.5 (dd, J ) 30, 27 Hz), 64.8, 43.5, 25.9, 22.8, 8.6 (2C),
6.4; HRMS for C22H21F2N7O3 (M + H)+ calcd 470.1752, found
470.1756.
5-(6-(5-(Cyclopropylmethyl)-1,3,4-oxadiazol-2-yl)-5-iso-
propylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-2,4-difluoro-
N-methoxybenzamide (43): off-white solid; Anal. RP-HPLC
tR ) 3.50 min (column B, purity 98%); 1H NMR (400 MHz,
CDCl3) δ 9.31-9.26 (m, 1H), 9.13 (bs, 1H), 8.07-7.93 (m, 2H),
7.46 (bs, 1H), 6.97 (t, 1H, J ) 10.2 Hz), 4.21-4.17 (m, 1H),
3.85 (s, 3H), 2.77 (d, 2H, J ) 7.1 Hz), 1.49 (d, 6H, J ) 7.1 Hz),
1.17-1.09 (m, 1H), 0.61-0.56 (m, 2H), 0.31-0.27 (m, 2H); 13
C
NMR (100 MHz, DMSO-d6) δ 166.2, 161.5, 161.2, 160.1, 156.5
(dd, J ) 250, 10 Hz), 154.5 (dd, J ) 245, 10 Hz), 153.5, 147.5,
129.9, 127.0, 124.5 (d, J ) 10 Hz), 119.9, 117.8, 116.3 (d, J )
15 Hz), 106.9, 105.1 (t, J ) 30 Hz), 63.7, 29.5, 25.1, 22.5, 21.4,
8.5, 4.8 (2C); HRMS for C23H23F2N7O3 (M + H)+ calcd 484.1908,
found 484.1904.
5-(6-(5-(Difluoromethyl)-1,3,4-oxadiazol-2-yl)-5-isopro-
pylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-2,4-difluoro-N-
methoxybenzamide (44): off-white solid; Anal. RP-HPLC tR
) 3.78 min (column B, purity 98%); 1H NMR (400 MHz, CD3-
OD) δ 8.13 (s, 1H), 7.73-7.68 (m, 1H), 7.59 (s, 1H), 7.23 (t,
1H, J ) 10.4 Hz), 7.13 (t, 1H, J ) 50 Hz), 4.11-4.05 (m, 1H),
3.73 (s, 3H), 1.42 (d, 6H, J ) 7.1 Hz); 13C NMR (100 MHz,
DMSO-d6) δ 162.9, 160.7, 157.7, 155.8 (dd, J ) 251, 14 Hz),
155.1 (dd, J ) 251, 14 Hz), 141.4, 139.5, 131.9 (d, J ) 15 Hz),
130.5, 124.6, 122.4, 118.6 (d, J ) 15 Hz), 116.7, 107.0 (t, J )
238 Hz), 106.0 (t, J ) 30 Hz), 105.5, 63.7, 25.2, 21.3, 21.1;
HRMS for C20H17F4N7O3 (M + H)+ calcd 480.1407, found
480.1410. Anal. (C20H17F4N7O3) C, H, N.
2,4-Difluoro-5-(5-isopropyl-6-(5-(trifluoromethyl)-1,3,4-
oxadiazol-2-yl)-pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-
methoxybenzamide (45): off-white solid; Anal. RP-HPLC tR
) 3.81 min (column B, purity 98%); 1H NMR (400 MHz, CD3-
OD) δ 8.09 (s, 1H), 7.71-7.67 (m, 1H), 7.56 (s, 1H), 7.24 (t,
1H, J ) 10.1 Hz), 4.35 (hep, 1H, J ) 7.2 Hz), 3.80 (s, 3H),
1.51 (d, 6H, J ) 7.2 Hz); 13C NMR (100 MHz, DMSO-d6) δ
163.7, 160.5, 160.1, 156.0 (dd, J ) 255, 17 Hz), 155.6 (dd, J )
255, 17 Hz), 153.5, 153.1, 143.3, 130.5, 122.8, 122.2, 118.6 (d,
J ) 20 Hz), 118.0 (q, J ) 274 Hz), 116.7, 116.3, 106.0 (t, J )
28 Hz), 63.7, 25.3, 21.3 (2C); HRMS for C20H16F5N7O3 (M +
H)+ calcd 498.1313, found 498.1297.
A mixture of 6-(5-(dimethylamino)-1,3,4-oxadiazol-2-yl)-5-
isopropylpyrrolo[2,1-f][1,2,4]triazin-4(3H)-one (91 mg, 0.32
mmol) in POCl3 (3 mL) was heated to 120 °C for 5 h. The excess
POCl3 was removed under reduced pressure and the residue
was coevaporated with toluene. The residue was dissolved in
MeCN (5 mL) and treated with 5-amino-2,4-difluoro-N-meth-
oxybenzamide (63 mg, 0.31 mmol). The reaction mixture was
stirred at room temperature for 16 h, the solvent was removed
under reduced pressure, and the residue was purified by
preparative HPLC to afford 47 (81 mg, 55%) as an off white
solid: Anal. RP-HPLC tR ) 1.50 min (column A, purity 99%);
1H NMR (400 MHz, CD3OD) δ 7.78 (s, 1H), 7.59-7.55 (m, 1H),
7.39 (m, 1H), 7.07 (t, 1H, J ) 10.1 Hz), 4.09-4.01 (m, 1H),
3.68 (s, 3H), 3.06 (s, 6H), 1.32 (d, 6H, J ) 7.1 Hz); HRMS for
C21H22F2N8O3 (M + H)+ calcd 473.1805, found 473.1810.
2,4-Difluoro-5-(5-isopropyl-6-(5-(2,2,2-trifluoroethyl)-
1,3,4-oxadiazol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-
N-methoxybenzamide (46): pale yellow oil; Anal. RP-HPLC
tR ) 1.71 min (column A, purity 99%); 1H NMR (400 MHz,
CDCl3) δ 9.17-9.14 (m, 2H), 8.03-7.97 (m, 2H), 7.45 (bs, 1H),
7.00 (t, 1H, J ) 10.3 Hz), 4.19-4.14 (m, 1H), 3.86 (s, 3H), 3.79
(q, 2H, J ) 9.6 Hz), 1.50 (d, 6H, J ) 7.2 Hz); MS (ESI) m/z
512 (M + H)+.
5-(6-(5-((Dimethylamino)methyl)-1,3,4-oxadiazol-2-yl)-
5-isopropylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-2,4-di-
fluoro-N-methoxybenzamide, Trifluoroacetic Acid Salt
(48): pale yellow oil; Anal. RP-HPLC tR ) 1.23 min (column
A, purity 90%); 1H NMR (400 MHz, CD3OD) δ 8.01 (s, 1H),
5-(6-(5-(Difluoro(methylsulfonyl)methyl)-1,3,4-oxadia-
zol-2-yl)-5-isopropylpyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-
2,4-difluoro-N-methoxybenzamide (50). To a solution of 49
(0.89 g, 1.7 mmol) in THF (100 mL) at -78 °C was slowly
added 1 N lithium bis(trimethylsilyl)amide in THF (6.9 mL,

4004 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
6.9 mmol). The reaction mixture was stirred for 10 min at -78
°C and then warmed to -40 °C. A solution of N-fluorobenzene
sulfonamide (1.4 g, 4.3 mmol) in THF (10 mL) was added
dropwise to the reaction mixture and the resulting solution
was stirred for 1 h at -30 to -40 °C. The reaction was
quenched by the addition of cold saturated aqueous NH4Cl
solution (100 mL), and the mixture was extracted with EtOAc
(3 × 100 mL). The EtOAc portions were dried (Na2SO4) and
concentrated in vacuo, and the residue was purified by flash
chromatography (SiO2, 0-2% MeOH/CH2Cl2 gradient elution)
to give 50 (0.63 g, 65%) as a white solid: Anal. RP-HPLC tR )
3.33 min (column B, purity 97%); 1H NMR (400 MHz, CDCl3)
δ 9.09-9.03 (m, 2H), 8.04 (s, 1H), 7.93 (s, 1H), 7.50 (bs, 1H),
6.91 (t, 1H, J ) 10.3 Hz), 4.08-4.01 (m, 1H), 3.78 (s, 3H), 3.16
(s, 3H), 1.43 (d, 6H, J ) 7.0 Hz); 13C NMR (100 MHz, CDCl3
with one drop CD3OD) δ 164.4, 161.2, 155.6 (dd, J ) 256, 14
Hz), 154.9 (dd, J ) 256, 14 Hz), 153.2 (t, J ) 30 Hz), 133.0,
127.5, 126.0 (2C), 121.5, 116.2, 116.1, 113.8 (t, J ) 288 Hz),
105.4, 104.6 (t, J ) 28 Hz), 63.6, 35.3, 25.5, 21.0 (2C); HRMS
for C21H19F4N7O5S (M + H)+ calcd 558.1165, found 558.1163.
Anal. (C21H19F4N7O5S) C, H, N.
Tris, pH 7.0, 25 µg/mL BSA, 1.5 mM MnCl2, 0.5 mM dithio-
threitol). The Flk-1 kinase reactions consisted of 10 ng of GST-
Flk-1 enzyme, 75 µg/mL substrate, 1 µM ATP, and 0.04 µCi
[γ-33P]ATP in 50 µL total reaction volume (kinase buffer: 20
mM Tris, pH 7.0, 25 µg/mL BSA, 4 mM MnCl2, 0.5 mM
dithiothreitol). The FGFR-1 kinase reactions consisted of 10
ng of GST-FGFR-1 enzyme, 75 µg/mL substrate, 1 µM ATP,
and 0.04 µCi [γ-33P]ATP in 50 µL total reaction volume (kinase
buffer: 20 mM Tris, pH 7.0, 25 µg/mL BSA, 0.5 mM MnCl2,
0.5 mM MgCl2, and 0.5 mM dithiothreitol). The PDGFR-â
kinase reactions consisted of 10 ng GST-PDGFR-â enzyme, 180
µg/mL substrate, 1 µM ATP, and 0.04 µCi [γ-33P]ATP in 50
µL total reaction volume (kinase buffer: 16 mM HEPES, pH
7.0, 80 µg/mL BSA, 4 mM MnCl2, 120 mM NaCl, and 0.5 mM
dithiothreitol). In all cases, the reactions were incubated for
60 min at 27 °C and terminated with the addition of cold
trichloroacetic acid (TCA) to a final concentration of 15%.
For the IGF-1R kinase assay, reactions consisted of 125 ng
of GST-IGF-1R enzyme, 25 ng substrate, and 25 µM of [γ-33P]-
ATP in a final reaction volume of 50 µL (kinase buffer: 20
mM MOPS, pH 7.0, 0.1 mg/mL BSA, 5 mM MnCl2, and 0.1
mM dithiothreitol). The reactions were incubated at 27 °C for
60 min.
2,4-Difluoro-5-(5-isopropyl-6-(5-methyl-4H-1,2,4-triazol-
3-yl)pyrrolo[2,1-f][1,2,4]triazin-4-ylamino)-N-methoxy-
benzamide, Hydrochloride Salt (52). A mixture of 35 (24
mg, 0.10 mmol) and acetamidine hydrochloride (14 mg, 0.15
mmol) in DMF (1 mL) was heated at 120 °C for 1 h. The
reaction mixture was diluted with MeOH and purified by
preparative HPLC to give 51 (22 mg, 0.086 mmol, 86%) as a
white solid: Anal. RP-HPLC tR ) 1.71 min (column B, purity
For HER-2, the entire cytoplasmic sequence was expressed
without an affinity tag. The protein was partially purified by
ion-exchange chromatography on DEAE-Sepharose (Pharma-
cia Biotech) and was eluted with a buffer containing 0.3 M
NaCl. For both the HER-1 and HER-2 kinase assays, the
reactions consisted of 10 ng of GST-HER-1 or 150 ng of
partially purified HER-2 enzyme, 1.5 µM substrate, 1 µM ATP,
and 0.15 µCi [γ-33P]ATP in a total reaction volume of 50 µL
(kinase buffer: 50 mM Tris-HCl pH 7.7, 0.1 mg/mL BSA, 10
mM MnCl2, and 2 mM dithiothreitol). The reactions were
incubated at 27 °C for 60 min and terminated by the addition
of 10 µL of stop buffer (2.5 mg/mL BSA and 0.3 M EDTA),
followed by 108 µL of a mixture of 3.5 mM ATP and 5% TCA.
The percent inhibition from the kinase assays was determined
by nonlinear regression analyses, and data were reported as
the inhibitory concentration required to achieve 50% inhibition
relative to control reactions (IC50).
1
97%); H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.90 (s,
1H), 4.21 (hep, 1H, J ) 7.2 Hz), 2.40 (s, 3H), 1.35 (d, 6H, J )
7.1 Hz); MS (ESI) m/z 259 (M + H)+.
The triazole 51 (22 mg, 0.086 mmol) was treated with POCl3
(1 mL) and the reaction mixture was stirred at 120 °C for 5 h.
The excess POCl3 was removed under reduced pressure and
the residue was coevaporated with toluene. The residue was
dissolved in 2-propanol and stirred with N-methoxyl-5-amino-
2,4-difluorobenzamide (20 mg, 0.10 mmol) at 50 °C for 1 h.
The solvent was removed in vacuo and the residue was purified
by preparative HPLC. The product was converted to the
hydrochloride salt by treatment with aqueous 1 N HCl followed
by lyophilization to afford 52 (27 mg, 71%) as a white solid:
Anal. RP-HPLC tR ) 3.17 min (column B, purity 98%); 1H NMR
(400 MHz, CD3OD) δ 7.88 (s, 1H), 7.69-7.63 (m, 1H), 7.56 (s,
1H), 7.22 (t, 1H, J ) 10.2 Hz), 4.10 (hep, 1H, J ) 7.2 Hz), 3.82
(s, 3H), 2.74 (s, 3H), 1.43 (d, 6H, J ) 7.2 Hz); HRMS for
C20H20F2N8O2 (M + H)+ calcd 443.1742, found 443.1748.
In Vitro Kinase Assays. The enzyme inhibition studies
were carried out as previously described.23 Recombinant
proteins containing either the cytoplasmic domains of the
human receptor tyrosine kinases (VEGFR-2, Flk-1, FGFR-1,
PDGFR-â, IGF-1R, HER-1, HER-2) or the entire protein
sequence for the serine/threonine kinases (PKC and CDK2)
were expressed as n-terminal glutathione S-transferase (GST)
fusion proteins using the baculovirus expression vector system
in Sf9 cells. The resultant proteins were isolated by affinity
chromatography using glutathione-Sepharose (Pharmacia Bio-
tech). All enzymes were stored at -80 °C. The kinase assays
were performed in 96-well microtiter plates using the synthetic
polymer poly(Glu4/Tyr) (Sigma Chemicals) as a phosphoac-
ceptor substrate. TCA precipitates were collected onto GF/C
unifilter plates (Packard Instrument Co., Meriden, CT) using
a Filtermate universal harvester (Packard Instrument Co.),
and the filters were quantitated using a TopCount 96-well
liquid scintillation counter (Perkin-Elmer Life Sciences). Pro-
cedures for assessing activity of the representative nonreceptor
tyrosine kinase Lck and the serine-threonine kinases PKCR
and CDK2 have been previously described.34,35
The apparent Ki value for 50 was determined by global
fitting of background-corrected cpm data to a competitive
model versus ATP using GraFit software, version 5 (Erithacus
Software Ltd).
Cellular Proliferation Assays. Primary human umbilical
vein endothelial cells (HUVECs) were purchased from Clonet-
ics (catalog # CC-2519) and not used beyond passage 3 for the
mitogen-stimulated proliferation assays as previously de-
scribed.23 The concentrations of VEGF (Peprotech) and FGF
(Clonetics) used in the mitogen-stimulated HUVEC assays
were 8 and 80 ng/mL, respectively. The L2987 human lung
carcinoma cells were not used beyond passage 20 for serum-
stimulated proliferation assays. For compound assessment,
cells were grown in 100 µL of minimal growth medium (Cellgro
catalog # 10-040-CV) and 1.0% heat-inactivated fetal bovine
serum (Cellgro catalog # 10-040-CV) in 96-well collagen IV
coated plates (Bectin-Dickinson, catalog # 354429) at a density
of 2 × 103 per well in a 37 °C/5% CO2 environment. Twenty-
four hours later, serum was adjusted to 10%, and test
compounds at various dilutions were added to each well in a
final volume of minimal growth media that contains 10%
serum. Forty-eight hours later, 0.5 µCi of [3H]thymidine
(Amersham catalog # TRK120) was added in a volume of 20
µL of minimal media for 24 h. Plates were washed once in PBS.
Upon removal of PBS, Trypsin (Cellgro catalog numbers 21-
031-CV and 25-054-CI, respectively) was added to cells which
were subsequently harvested onto glass-fiber filters (Perkin-
Elmer-Life-Sciences catalog # 1450-525) using an automated
harvestor (Brandel Model# MWX RI-192TI). Incorporated
tritium was quantified using a â-counter (Wallac Microbeta).
Dose-response curves were generated to determine the IC50
value, which is defined as the concentration of drug required
to inhibit 50% of tritium incorporation when compared to
untreated serum-stimulated cells.
For the VEGFR-2, Flk-1, FGFR-1, and PDGFR-â kinase
assays, compounds were dissolved in DMSO and diluted with
water/10% DMSO to a final DMSO concentration of 2%. The
VEGFR-2 kinase reactions consisted of 8 ng of GST-VEGFR-2
enzyme, 75 µg/mL substrate, 1 µM ATP, and 0.04 µCi [γ-33P]-
ATP in 50 µL total reaction volume (kinase buffer: 20 mM

VEGFR-2 and FGFR-1 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 4005
Metabolic Stability. The compound was incubated with
human liver microsomes purchased from In Vitro Technologies
(Baltimore, MD) and pooled from 10 individual donors. The
rates of oxidative metabolism were measured under the
following conditions: compound (substrate) at 3 or 10 µM final
concentration, final microsomal protein concentration of ap-
proximately 1 mg/mL, 1 mM NADPH, and 56 mM of pH 7.4
potassium phosphate buffer. Incubations were performed at
37 °C and were initiated by the addition of the substrate.
Incubations were quenched by the addition of one volume of
acetonitrile after 10 min. Samples were then analyzed for the
parent compound by LC-MS. The rate of metabolism was
calculated by determining the nanomoles of parent compound
oxidized and dividing it by the time of incubation and the
milligrams of protein.
mice, n ) 3 for human) obtained from Bioreclamation Inc.
(Hicksville, NY). Compound 50 (1 mM) in acetonitrile was
added to serum at a ratio of 1:100 to give a final concentration
of 10 µM. Serum samples were dialyzed against 134 mM
phosphate buffer (pH 7.4). The Micro-Equilibrium Dialyzer
(500 µL chamber volume, Amika Corp., Holliston, MA) con-
taining spiked serum was incubated in a shaking water bath
maintained at 37 °C for 4 h. A 10 000 molecular weight cutoff
dialysis membrane (Amika Corp., Holliston, MA) was used.
All experiments were carried out in triplicate. Aliquots of
buffer and serum were taken at 4 h, and analyzed by LC-
MS/MS. Compound 50 was stable under these conditions over
the 4 h incubation period in mouse and human serum.
From each dialysis cell, the free and bound drug percentages
were calculated as follows:
Cytochrome P450 Assay. The ability of compounds to
inhibit the major human cytochrome P450s (CYPs) responsible
for drug metabolism was evaluated in vitro using recombinant
human CYP isoforms. The inhibition of cDNA-derived CYP
enzymes prepared from baculovirus-infected insect cells was
measured using either 3-cyano-7-ethoxycoumarin (CYP1A2,
CYP2C19), 7-methoxy-4-trifluoromethylcoumarin (CYP2C9),
or 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-me-
thylcoumarin (CYP2D6) as the substrates. CYP3A4 inhibition
was evaluated with two substrates: 7-benzyloxy-4-trifluoro-
methylcoumarin (BFC) and resorufin benzyl ether (BzRes). A
single concentration of each model substrate (at approximately
the apparent Km, with the exception of BFC, which was tested
below the apparent Km) and multiple concentrations of the test
compounds, separated by approximately 1/2 log units, were
tested in duplicate. Metabolism of the model substrates was
assayed by the production of 7-hydroxy-3-cyanocoumarin, 3-[2-
(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin, 7-hy-
droxy-4-trifluoromethylcoumarin, or resorufin and measured
via fluorescence detection. Assays were conducted in 96-well
microtiter plates in the presence of an NADPH-generating
system. Positive control samples were included in these
studies. The positive control values were within the historical
range for all the assays. The IC50 values were calculated
utilizing XLfit curve-fitting software.
Pharmacokinetic Parameters Obtained in Mice. In
these 4 h oral exposure studies, a single-dose of 50 mg/kg of
compounds 24-26, 37, 44, 49, and 50 (10 mg/kg) was delivered
as a solution in 70% PEG400/10% ethanol/20% water by oral
gavage to fasted adult male Balb/C mice (n ) 3 per compound).
Three serum samples were collected from each mouse at 0.5,
1, and 4 h time points following oral dosing. The first two
samples were obtained by retro-orbital bleed (∼100 µL/ 20-
25 g mouse) and the third sample by cardiac puncture. The
blood samples were allowed to clot on ice and centrifuged and
then the serum was harvested. Plasma samples were stored
at -20 °C prior to analysis. The plasma samples were analyzed
for parent compound via HPLC-coupled tandem mass spec-
trometry (LC-MS/MS).
% free ) 100 × (concentration in buffer)/
(concentration in serum)
% bound ) 100 - % free
In Vivo Antitumor Activity in the Subcutaneously
Implanted L2987 Xenograft Model in Nude Mice. Female
Balb/C athymic (nu+/nu+) mice, 6-8 weeks old, were obtained
from Sprague-Dawley Co. (Indianapolis, IN). Animals were
provided with food and water ad libitum and housed five per
cage. Mice were maintained in accordance with Bristol-Myers
Squibb’s Institutional Animal Care and Use Committee in
accordance with the American Association for Accreditation
of Laboratory Animal Care (AAALAC) guidelines for the
humane treatment and care of laboratory mice.
L2987 tumor fragments42 maintained by serial passage in
vivo were implanted subcutaneously in the hind flank using
an 18 g trocar. Approximately 2 weeks postimplant, when
tumor sizes reached 100-150 mm3, oral dosing was initiated
using gavage needles with either compound at the indicated
concentrations or vehicle (7:1:2 PEG400/EtOH/H2O) in the
control group. Tumor growth was assessed twice weekly by
vernier caliper measurement. Group sizes were n ) 8 or 9.
Treatments resulting in greater than 20% lethality and/or
20% body weight loss were considered toxic. Antitumor activity
was determined by calculating the maximum percent tumor
growth inhibition (TGI) of treated animals at the indicated
time points using the formula
%TGI ) {(Ct - Tt)/(Ct-C0)} × 100
where Ct ) the median tumor volume (mm3) of vehicle-treated
control (C) mice at time t. Tt ) median tumor volume of treated
(T) mice at time t. C0 is the median tumor volume of control
mice at time 0. Activity is defined as a continuous %TGI >
50% for at least one tumor volume doubling time after the start
of drug treatment.
To evaluate the oral bioavailability of compound 50 in male
Balb/C mice, a single dose was delivered as a solution in 30%
PEG 400/10% ethanol/60% water by either tail vein injection
(iv, 5 mg/kg) or by oral gavage (po, 10 mg/kg). The mice were
fasted overnight prior to dosing and fed 4 h post dose. A total
of 18 mice were used in the study (n ) 9 each for iv and po
groups). Three serum samples were collected from each mouse,
the first two samples by retro-orbital bleed (∼100 µL/ 20-25
g mouse) and the third sample by cardiac puncture. Blood
samples were collected at 0.5, 0.5, 1, 3, 6, 8, 10, 12, and 24 h
time points following iv dosing and at 0.25, 0.5, 1, 3, 6, 8, 10,
12, and 24 h following oral dosing. Blood samples were allowed
to clot on ice and centrifuged, and then serum was harvested.
Plasma samples were stored at -20 °C until analysis. Con-
centrations of parent compound were later determined by LC-
MS/MS. Composite serum concentration-time profiles were
constructed for pharmacokinetic analysis.
Acknowledgment. The authors wish to thank John
Hynes for his seminal contributions on the hydroxam-
ate-based pyrrolo[2,1-f][1,2,4]triazines; Amy Camuso,
Steven Dzwonczyk, Kurt Gregor, Stephanie Kut, Aixin
Li, and Chiang Yu for conducting the kinase selectivity
assays; Hilary Gray for protein expression and purifica-
tion; Mark Witmer for assistance with the enzyme
kinetics; Victor Cardenas for informatics support; Brian
Fink for helpful discussions; and the Discovery Analyti-
cal Sciences Department for obtaining high-resolution
MS analyses.
Supporting Information Available: A general procedure
for the synthesis of the C6-carbamate library, a table of
pertinent data for the C6-carbamate analogues, and a table
of combustion analysis or HPLC analysis data for key com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Serum Protein Binding of Compound 50. The extent
of protein binding of compound 50 was determined in mouse
and human sera using the equilibrium dialysis method. All
experiments were carried out using pooled serum (n ) 10 for

4006 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Borzilleri et al.
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